Mild Hyperthermic Conditions Research Articles

Thermally Targeted p50 Peptide Inhibits Proliferation and Induces Apoptosis of Breast Cancer Cell Lines.

The application of rationally designed therapeutic peptides (TP) may improve outcomes in cancer treatment. These peptides hold the potential to directly target proliferative pathways and stimulate cell arrest or death pathways. Elastin-like polypeptide (ELP) is an elastin derived biopolymer that undergoes a thermally mediated phase transition. This study employs p50, a nuclear localization sequence derived peptide that inhibits the activation of NFκB and is implicated in cancer cell survival and metastasis. In order to effectively delivery p50, it is conjugated to SynB1-ELP1, a thermally responsive macromolecular carrier. By applying an external heat source, mild hyperthermic conditions (41 °C) induce aggregation and therefore can be used to specifically target ELP to solid tumors in cancer therapy. The addition of a cell penetrating peptide (CPP) to the N-terminus of the macromolecular carrier enhances the cellular uptake and directs the subcellular localization of the bioactive peptide. The novel TP, p50, inhibits proliferation and induces apoptosis of breast cancer cells by blocking the intranuclear import of NFκB. By expanding the repertoire of oncogenic targets, CPPs, and ELP carrier sizes, ELP-based polypeptides may be modulated to optimize the delivery of these novel therapies and allow for the flexibility to create individualized cancer therapies.

Isolated limb perfusion for soft-tissue sarcoma and regional melanoma.

10579 Background: Isolated limb perfusion (ILP) represents a treatment option for locally advanced melanoma and sarcoma confined to a limb. The advantage of this approach is to deliver high-dose regional chemotherapy without serious systemic effects. However, the ILP technique involves a complex and invasive operative procedure, requiring accurate monitoring to avoid major local toxicity. Methods: From November 2004 to December 2011, 58 patients underwent IPL for unresectable soft tissu sarcoma (STS= 34) and advanced in-transit melanoma (n=24). IPLs were performed at mild hyperthermic conditions with 1-2 mg of TNF and 40-80 mg of melphalan (M) for arm and leg perfusions, respectively. The response rate, disease free intervals, overall survival, toxicity and limb salvage rate were evaluated. Results: Median age was 68 years (range: 29-91 years), with 58% of women. For sarcoma patients, median tumor size was 60 mm, 16 patients (47%) had a high grade STS. Twenty-one patients (61%) received IPL before definitive surgery. Eight patients finally underwent amputation, giving a long-term limb salvage of 77%. The overall response rate was 73.5% (Complete response rate 14.7%, partial response rate 58.8 %). For melanoma patients, 9 (38%) had an AJCC stage III disease, the median thickness of the primary tumor was 3.5 mm. A complete response was obtained in 21% of patients while 54% exhibited a partial response. The local and metastatic recurrence rates were similar between sarcoma and melanoma patients (41% and 33% respectively). All but one of the patients with non-operated sarcoma presented a local or metastatic relapse.There was no mortality and no systemic toxicity. Regional toxicity (Wieberdink scale) was: grade I (no reaction) 53 %, II (erythema, oedema) 34%, III (blistering) 8% and IV 3%. The median local relapse-free survival was 40 months in sarcoma group (26.6 months for non operated patients) and 10 months in melanoma one. The overall 3-years survival rate was 44% for sarcoma and 25% for melanoma patients. Conclusions: ILP induces a high tumour response rate, leads to a high limb salvage rate but is associated with an important recurrence rate. It provides a limb salvage alternative to amputation when local control is necessary.

Long-Term Results of Tumor Necrosis Factor α– and Melphalan-Based Isolated Limb Perfusion in Locally Advanced Extremity Soft Tissue Sarcomas

Because there is no survival benefit of amputation for extremity soft tissue sarcomas (STSs), limb-sparing surgery has become the gold standard. Tumor size reduction by induction therapy to render nonresectable tumors resectable or facilitate function-preserving surgery can be achieved by tumor necrosis factor α (TNF) -based and melphalan-based isolated limb perfusion (TM-ILP). This study reports the long-term results of 231 TM-ILPs for locally advanced extremity STS. We analyzed 231 TM-ILPs in 208 consecutive patients (1991 to 2005), who were all candidates for functional or anatomic amputation for locally advanced extremity STS. All patients had a potential follow-up of up to 5 years. TM-ILP was performed under mild hyperthermic conditions with 1 to 4 mg of TNF and 10 to 13 mg/L of limb-volume melphalan. Almost all patients (85%) had intermediate- or high-grade tumors. The overall response rate (ORR) was 71% (complete response, 18%; partial response, 53%). Multifocal sarcomas had a significantly better ORR of 83% (P = .008). The local recurrence rate was 30% (n = 70); local recurrence rates were highest for multifocal tumors (54%; P = .001) and after previous radiotherapy (54%; P < .001). Five-year overall survival rate was 42%. Survival was poorest in patients with large tumors (P = .01) and with leiomyosarcomas (P < .001). Limb salvage rate was 81%. We demonstrated that TM-ILP results in a limb salvage rate of 81% in patients with locally advanced extremity STS who would otherwise have undergone amputation. Whenever an amputation is deemed necessary to obtain local control of an extremity STS, TM-ILP should be considered.

20 Years Experience of TNF-Based Isolated Limb Perfusion for In-Transit Melanoma Metastases: TNF Dose Matters

BackgroundApproximately 5–8% of melanoma patients will develop in-transit metastases (IT-mets). Tumor necrosis factor-α (TNF) and melphalan-based isolated limb perfusion (TM-ILP) is an attractive treatment modality in melanoma patients with multiple IT-mets. This study reports on a 20 years experience and outlines the evolution and major changes since the introduction of TNF in ILP.MethodsA total of 167 TM-ILPs were performed in 148 patients, between 1991 and 2009. TM-ILPs were performed at high doses of TNF (3–4 mg) from 1991 to 2004 (n = 99) and at low doses of TNF (1–2 mg) from 2004 to 2009 (n = 68) under mild hyperthermic conditions (38°C–39.5°C.). Melphalan doses were unchanged at 10–13 mg/l (leg and arm, respectively). Characteristics for the 167 ILPs were: 81 stage IIIB, 65 stage IIIC, and 21 stage IV disease.ResultsThe overall response rate was 89% (n = 148). (Complete response [CR] = 61%; partial response [PR] = 28%). CR rates correlated with stage (P = .001) and with high-dose vs. low-dose TNF (70% vs. 49%; P < .006). High-dose TNF prolonged local control (median 16 months vs. 11 months; P = .076). Survival was not influenced by TNF dose. CR after ILP and number of lesions also correlated with local progression-free interval. Overall survival did correlate with stage of disease (P < .001), size of the lesions (P = .001), and a CR (P < .001).ConclusionsThis 2-decade single-center experience demonstrates that TM-ILP is a safe and effective treatment modality for melanoma patients with multiple IT-mets. Higher dose of TNF was associated with significantly higher CR rates and prolonged local control without an effect on overall survival.

Technique and outcomes of isolated limb infusion for locally advanced malignant melanoma — A radiological perspective

Isolated limb infusion (ILI) is a novel, minimally invasive technique for delivering high-dose regional chemotherapy in patients with recurrent and in-transit melanoma. The aim of this study was to review our single-centre experience in treating eleven patients. We emphasize the role of radiologists in setting up this service, including pre-treatment workup and placement of vascular catheters. A retrospective analysis of 11 patients who underwent 12 procedures between 2005 and 2009 was performed. Pre-procedural staging computed tomography (CT), CT angiography, and duplex studies were performed. All patients received a cytotoxic combination of melphalan and actinomycin-D via radiologically placed arterial and venous catheters in the affected limb under mild hyperthermic conditions. The outcome measures include response rates, limb toxicity, complications, and survival. All patients were female with a mean age of 72 years. Three patients had American Joint Committee on Cancer (AJCC) stage IIIB melanoma, seven had stage IIIC melanoma, and one had a stage IIIB Merkel cell tumour. Complete response was seen in five patients (46%), partial response in four (36%), and progressive disease in two (18%). One patient developed grade 4 toxicity requiring a fasciotomy and another experienced systemic toxicity. These outcomes are comparable to previous studies and shows that ILI is effective in locoregional control of unresectable melanoma. It is a relatively safe procedure but not without risk. Our experience shows the importance of radiological input to ensure safe and effective delivery of services.

Abstract 2659: A thermally targeted carrier of p50 NLS peptide inhibits cancer cell proliferation by preventing NFκB nuclear translocation

Abstract The application of rationally designed therapeutic peptides may improve outcomes in cancer treatment. These peptides hold the potential to directly target proliferative pathways and to inhibit their over-expression. The NFκB survival pathway is constitutively active in many cancers and plays a role in evading apoptosis. A p50 NLS-derived peptide has been shown to bind to the nuclear receptor and prevent NFκB translocation into the nucleus. This 10 amino acid peptide thus inhibits the transcriptional activation of NFκB's cellular targets that are implicated in cancer cell survival and metastasis. By conjugating the p50 derived peptide to a thermally targeted carrier and a cell penetrating peptide, the specificity and stability of peptide delivery can be enhanced. The addition of a cell penetrating peptide, SynB1, to the N-terminus of ELP enhances the cellular uptake of the conjugated peptide, SynB1-ELP1-p50. Elastin like polypeptide (ELP) is an elastin derived biopolymer that undergoes a thermally-mediated phase transition. By applying an external heat source, mild hyperthermic conditions (41°C) can induce aggregation and therefore can be used to specifically target ELP to solid tumors in cancer therapy. Uptake studies indicate that hyperthermia increases the accumulation of flourescein-labeled SynB1-ELP1-p50 in MCF-7 cells by 3 fold as compared to SynB1-ELP1-p50 treatment without heat. In addition to providing a level of targeting specificity, as a macromolecule, ELP may increase the plasma stability of small therapeutic peptides, like p50, and therefore creates the potential for viable delivery in vivo. Preliminary in vitro studies have demonstrated that two 1-hour treatments with 20μM of SynB1-ELP1-p50 inhibits cell proliferation by 20% and in combination with hyperthermia by 40%, in both MCF-7 and HeLa cell lines. No inhibitory effect was seen when the carrier control, SynB1-ELP1, was used. Confocal microscopy studies illustrate the predominantly cytoplasmic localization of rhodamine labeled SynB1-ELP1-p50 with some localization at the nuclear periphery. Immunofluorescence studies suggest that SynB1-ELP1-p50 may attenuate the nuclear translocation of NFκB following stimulation with TNF-α. Future studies will elucidate the anti-proliferative mechanisms of this conjugated peptide and quantify the inhibition of nuclear translocation of NFκB following treatment with SynB1-ELP1-p50. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2659.

Factors Predictive of Acute Regional Toxicity After Isolated Limb Infusion with Melphalan and Actinomycin D in Melanoma Patients

Isolated limb infusion (ILI) with cytotoxic drugs is a low-flow isolated limb perfusion (ILP) performed via percutaneous catheters without oxygenation to treat metastatic melanoma confined to a limb. Response rates and duration of response following ILI are similar to those after ILP. Previously we have shown that more significant limb toxicity is not associated with a higher response rate or improved patient outcome. In this study we sought to determine factors predicting toxicity following ILI. From our prospective database 185 patients with advanced metastatic melanoma of the limb treated with a single ILI between 1992 and 2007 were identified. In all patients a cytotoxic combination of melphalan and actinomycin D was used. Drug circulation time was 20-30 min under mild hyperthermic conditions (38-39 degrees C). Limb toxicity was assessed using the Wieberdink scale. The average patient age was 74 years (range 29-93 years) and 62% were female. Most patients (134/185) had MD Anderson stage III disease (satellites and in-transit metastases). Toxicity grade I (no reaction) occurred in 3 patients, grade II (slight erythema and edema) in 105 patients, grade III (considerable erythema and edema +/- blistering) in 72 patients, and grade IV (threatened or actual compartment syndrome) in 5 patients. No patient developed grade V toxicity (requiring amputation). On univariate analysis high peak and high final melphalan concentrations were found to be predictive factors for grade III/IV limb toxicity as well as the area under the curve of the melphalan concentration. Surprisingly, a greater rise in the CO(2) level during the procedure was associated with lower toxicity in the univariate analysis. Increased serum creatine phosphokinase (CK) postoperatively was related to higher toxicity score. In the multivariate analysis high final melphalan concentration and shorter tourniquet time were independent predictive risk factors for developing grade III/IV limb toxicity. ILI is a safe alternative to the more invasive and laborious ILP technique to treat melanoma confined to a limb. Regional acute toxicity following ILI is mild to moderate in most patients. Based on the predictive factors found in this series, altering melphalan dose and tourniquet time may allow further reductions in post-ILI toxicity without compromising effectiveness.

Characterization of the Effects and Potential Mechanisms Leading to Increased Megakaryocytic Differentiation Under Mild Hyperthermia

The physical culture parameters have important influences on the proliferation and differentiation fate of hematopoietic stem cells. Recently, we have demonstrated that CD34+ cord blood (CB) cells undergo accelerated and increased megakaryocyte (Mk) differentiation when incubated under mild hyperthermic conditions (i.e., 39 degrees C). In this study, we investigated in detail the impacts of mild hyperthermia on Mk differentiation and maturation, and explored potential mechanisms responsible for these phenomena. Our results demonstrate that the qualitative and quantitative effects on Mk differentiation at 39 degrees C appear rapidly within 7 days, and that early transient culture at 39 degrees C led to even greater Mk yields (p<0.03). Surprisingly, cell viability was only found to be significantly reduced in the early stages of culture, suggesting that CB cells are able with time to acclimatize themselves to 39 degrees C. Although mild hyperthermia accelerated differentiation and maturation of CB-derived Mks, it failed to promote their polyploidization further but rather led to a small reduction in the proportion of polyploid Mks (p=0.01). Conversely, gene arrays analysis demonstrated that Mks derived at 39 degrees C have a normal gene expression program consistent with an advanced maturation state. Finally, two independent mechanisms that could account for the accelerated Mk differentiation were investigated. Our results suggest that the accelerated and increased Mk differentiation induced by mild hyperthermia is not mediated by cell-secreted factors but could perhaps be mediated by the increased expression of Mk transcription factors.

Outcomes Following Isolated Limb Infusion for Melanoma. A 14-Year Experience

Isolated limb infusion (ILI) is a minimally invasive technique for delivering regional chemotherapy in patients with advanced and metastatic melanoma confined to a limb. It is essentially a low-flow isolated limb perfusion (ILP) performed via percutaneous catheters without oxygenation. From our prospective database 185 patients with advanced metastatic melanoma of the limb treated with a single ILI between 1993 and 2007 were identified. In all patients a cytotoxic drug combination of melphalan and actinomycin-D was used. Drug circulation time was 20-30 min under mild hyperthermic conditions (38-39 degrees C). The majority of patients (62%) were female. Their average age was 74 years (range 29-93 years). Most patients had MD Anderson stage III disease (134/185). The overall response rate was 84% [complete response (CR) rate 38%, partial response rate 46%]. Median response duration was 13 months (22 months for patients with CR; P = 0.01). Median follow-up was 20 months and median survival was 38 months. In those patients with a CR, the median survival was 53 months (P = 0.005). CR rate and survival time decreased with increasing stage of disease. On multivariate analysis significant factors for a favorable outcome were achievement of CR, stage of disease, thickness of primary melanoma, the CO(2 )level in the isolated circuit, and a Wieberdink limb toxicity score of III (considerable erythema and edema). The response rates and duration of response after ILI are comparable to those achieved by conventional ILP. ILI is a minimally invasive alternative to the much more complex and morbid conventional ILP technique for patients with advanced metastatic melanoma confined to a limb.

Outcome and prognostic factor analysis of 217 consecutive isolated limb perfusions with tumor necrosis factor‐α and melphalan for limb‐threatening soft tissue sarcoma

Extensive and mutilating surgery is often required for locally advanced soft tissue sarcoma (STS) of the limb. As it has become apparent that amputation for STS does not improve survival rates, the interest in limb-preserving approaches has increased. Isolated limb perfusion (ILP) with tumor necrosis factor-alpha (TNF) and melphalan is successful in providing local tumor control and enables limb-preserving surgery in a majority of cases. A mature, large, single-institution experience with 217 consecutive ILPs for STS of the extremity is reported. At a prospectively maintained database at a tertiary referral center, 217 ILPs were performed from July 1991 to July 2003 in 197 patients with locally advanced STS of the extremity. ILPs were performed at mild hyperthermic conditions with 1-4 mg of TNF and 10-13 mg/L limb-volume melphalan (M) for leg and arm perfusions, respectively. The overall response rate was 75%. Limb salvage was achieved in 87% of the perfused limbs. Median survival post-ILP was 57 months and prognostic factors for survival were Trojani grade of the tumor and ILP for single versus multiple STS. The procedure could be performed safely, with a perioperative mortality of 0.5% in all patients with no age limit (median age, 54 yrs; range, 12-91). Systemic and locoregional toxicity were modest and easily manageable. TNF+M-based ILP can provide limb salvage in a significant percentage of patients with locally advanced STS and has therefore gained a permanent place in the multimodality treatment of STS.

Isolated Limb Perfusion With Tumor Necrosis Factor and Melphalan Prevents Amputation in Patients With Multiple Sarcomas in Arm or Leg

Treatment for extremity soft tissue sarcoma (STS) has shifted in recent years from amputation to local wide excision combined with irradiation. For multiple sarcomas, this limb-sparing approach is often not possible. To avoid amputations, isolated limb perfusion (ILP) with tumor necrosis factor and melphalan is an attractive treatment option for patients with multiple extremity sarcomas. We investigated a prospective database at a tertiary referral institute. From July 1991 to July 2003, out of 217 ILPs, 64 ILPs were performed for either multifocal primary sarcomas or multiple sarcoma recurrences in 53 patients. All ILPs were performed under mild hyperthermic conditions by using 1 to 4 mg of tumor necrosis factor and 10 to 13 mg/L of limb volume for leg and arm perfusions, respectively. The overall response was 88%, with 42% complete response, 45% partial response, 11% no change, and 2% progressive disease. This response rate is significantly better than our experience in 153 locally advanced single-STS cases (88% vs. 69%). The toxicity of the procedure was mild to moderate in almost all cases; no treatment-related amputation had to be performed. The time to local recurrence was 29 months and differed significantly between multiple primary and multiple recurrent STS. The 5-year survival rate was 39%. Limb salvage was achieved in 45 (82%) of 55 treated limbs. In a group of patients who are uniformly candidates for amputation, ILP can achieve limb salvage in approximately four out of five patients. Because this treatment option provides excellent local control, it should be considered before an amputation is planned.

One Hundred Consecutive Isolated Limb Perfusions With TNF-α and Melphalan in Melanoma Patients With Multiple In-Transit Metastases

The aim of this study is to describe the experience with 100 TNF-based ILP for locally advanced melanoma and to determine prognostic factors for response, time to local progression, and survival. One hundred TNF-based ILPs were performed between 1991 and 2003 in 87 patients for whom local control by surgery of in-transit melanoma metastases was impossible. In total, 62 iliac, 33 femoral, and 5 axillary ILPs were performed in mild hyperthermic conditions with 2 to 4 mg of TNF and 10 to 13 mg of melphalan per liter of limb volume. Overall response was 95%, with 69% complete response, 26% partial response, and 5% no change. Complete response rate differed significantly for patients with IIIA disease versus IIIAB and IV. Local and systemic toxicity was mild to moderate in almost all cases, with no treatment-related death and one treatment-related amputation. Five-year overall survival was 32%; local progression occurred in 55% after a median of 16 months. In complete response patients, 5-year survival was 42% with local progression in 52% at a median of 22 months. Response rate and survival were significantly influenced by stage of disease; (local progression free) survival was influenced by response rate. TNF-based ILP results in excellent response rates in this patient population with unfavorable characteristics. Response on ILP predicts outcome in patients and reflects aggressiveness of the tumor.

Sweating responses during activation of the muscle metaboreflex in humans is altered by time of day

The aim of the present study was to test for a time-of-day effect on sweating responses to activation of the muscle metaboreflex. Eight male subjects each participated in two exercise sessions, one in the morning and one in the evening. Within each session there were two 60-s bouts of isometric handgrip (IHG) exercise at 50% maximal voluntary contraction. Prior to IHG, whole body warming by a water-perfused suit initiated mild sweating. The first bout of IHG exercise began at 06.00 hours (am) and 18.00 hours (pm). Blood circulation to the forearm was occluded for 120 s, beginning 5 s before the end of the second bout of IHG to activate the muscle metaboreflex. During both bouts of exercise, sweating rate (SR) on both the chest and right forearm significantly increased from the pre-exercise period in both am and pm sessions. SR rapidly decreased during first minute of recovery after the first bout of IHG exercise. However, during post-exercise ischaemia (PEI) after the second bout of IHG exercise, SR was maintained significantly above the pre-exercise level only in the pm session. The increases in SR on the chest and right forearm during PEI were significantly greater in the pm, than in the am, session. However, SR of the palm was not maintained during PEI. We conclude that under mild hyperthermic conditions, the sweating response in non-glabrous skin to activation of the muscle metaboreflex exhibits a time-of-day effect.

Prognostic factors for survival after isolated limb perfusion for malignant melanoma

Aims: Risk factors were determined for mortality within 1 year after isolated limb perfusion (ILP).Methods: All of 439 patients who underwent ILP for melanoma of the extremities were studied. Ninety percent of the patients had MD Anderson stage IIB or III disease at the time of ILP. ILP was performed with melphalan with or without TNFα under mild hyperthermic (38–40 °C) or normothermic (37–38 °C) conditions in 80% of the cases.Results: Sixty-nine patients died within this period, 64 of metastatic melanoma. The indication for ILP was an unresectable primary (n=3), a local recurrence (n=24) or adjuvant to excision of primary lesions (n=17) in patients with stage IIIB regional lymph node metastases. These patients or patients with stage IIIAB melanoma with satellites and/or in-transit metastases with regional lymph node metastases had a relative risk of 4.6 (95% CI 2.0–6.6) and 3.6 (95% CI 2.1–10) of dying within 1 year from ILP, respectively (p<0.001). In patients with stage IV disease (distant metastases), the relative risk was 22 (95% CI 3.8–127, p=0.001).Conclusion: Patients with advanced limb melanoma have an increased risk of death within 1 year after ILP when regional lymph node or distant metastases are present.

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells.

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.

Effects of incubation with liposomes at different temperatures on cultured melanoma cells (M14).

A melanoma cell line (M14) was used in order to investigate the effect of hyperthermia on the mechanisms of interaction between liposomes and cultured cells. The treatment was performed by adding different concentrations of multilamellar liposomes (L-alpha-dipalmitoylphosphatidylcholine, stearylamine and cholesterol in the ratio 7:2:1) to cell cultures which were then incubated at 37.0 or 41.5 degrees C for 2 h. The damage induced by liposome treatment in normothermia or hyperthermia was evaluated by determining cell survival and by electron microscopy. When different concentrations of liposomes were used, a dose-dependent impairment of cell survival was observed. An enhancement of the cytotoxic effect was observed when the treatment was performed at 41.5 degrees C. This effect went on even after 24 h from the end of the treatment, but the difference between cells treated in normothermia and hyperthermia was remarkably reduced. The mechanism of the liposome-plasma membrane interaction has been investigated by electron microscopy. Our observations demonstrated that the outer bilayer of the multilamellar liposomes was capable of fusing with the plasma membrane, inducing changes in its fluidity and molecular organization. Following this process the inner liposomal bilayers entered the cell. These effects seemed to be favoured when the treatment was performed under mild hyperthermic conditions, accounting for the synergic cytotoxic action displayed by the liposome-hyperthermia association.

Hyperthermia specifically inhibits bivalent chromosome disjunction in maturing mouse oocytes.

The effect of hyperthermia on mammalian oocyte maturation was studied by allowing preovulatory mouse oocytes to mature spontaneously for 17 h in vitro under controlled temperature conditions. At the end of culture, oocytes were screened for their maturation stage and for chromosome morphology and number. Mild hyperthermic conditions (38.5-40.0 degrees C) during maturation specifically disturbed the process of bivalent chromosome disjunction, but not other maturation steps, by blocking oocytes at the metaphase I stage and preventing cells from entering subsequent maturation steps. Some oocytes that had reached metaphase II under hyperthermic conditions had chromosome imbalance. Oocytes matured at 40.0 degrees C displayed chromosome morphological abnormalities, including altered sister chromatid separation and nucleus/nuclei formation, at a frequency significantly higher than oocytes matured at 37.0-39.0 degrees C. When incubation temperature was raised above 40.0 degrees C, increasing fractions of oocytes were inhibited from entering initial maturation steps. We conclude that hyperthermia during mammalian oocyte maturation specifically damages the process of bivalent chromosome disjunction and induces the appearance of chromosome structural defects and imbalance in unfertilized eggs.

In transit metastases of malignant melanoma treated by high dose rTNF alpha in combination with interferon-gamma and melphalan in isolation perfusion.

To increase the therapeutic efficacy of recombinant tumor necrosis factor alpha (rTNF alpha) and reduce the systemic side effects, a protocol was designed using isolation perfusion of the limbs with hyperthermia for in transit metastases of melanoma. A triple combination of high dose rTNF alpha + recombinant interferon-gamma (rIFN-gamma) + melphalan was chosen because of a synergistic anti-tumor effect of rTNF alpha with rIFN-gamma and of rTNF alpha with alkylating agents reported in the literature. Twenty-nine patients of mean age 60 years (range 22-82 years) entered the study after informed consent and received a total of 31 isolation perfusions with the triple combination. There were 24 women and 5 men with multiple progressive in transit melanoma metastases of the lower limb (stage IIIa or IIIab). rTNF alpha at the unique dose of 4 mg was injected as a bolus in the arterial line, under mild hyperthermic conditions (40 to 40.5 degrees C) for 90 minutes. rIFN-gamma was given subcutaneously on days -2 and -1 and in the perfusate, with rTNF alpha, at the dose of 0.2 mg. Melphalan was administered in the perfusate at dose giving a concentration of 40 micrograms/ml. In all the 31 isolation perfusions performed in the triple combination protocol, in order to prevent a septic shock-like syndrome which had been encountered in 2 patients treated outside this protocol for sarcoma and carcinoma, the patients received dopamine continuous infusion at 3 micrograms/kg/min from the start of isolation perfusion and for 48 hours, and only showed mild hypotension and very transient chills and temperature.(ABSTRACT TRUNCATED AT 250 WORDS)