Analysis of cell proliferation and cell death during in situ hyperthermic treatment of neoplastic cells: a case report of human non-Hodgkin lymphoma.

In this study we observed the effects in vivo of hyperthermic treatment on the cell kinetics (cell proliferation/cell death) in one case of human non-Hodgkin lymphoma, by analyzing the following morpho-cytochemical parameters: Acridine Orange fluorochromasia, mitotic index, proliferating cell nuclear antigen (PCNA) expression, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) labeling, and ultrastructure morphology. After two hyperthermic exposures there was a significant reduction of cell growth rate (e.g. mitotic and PCNA positive cells) and an increase in cell loss by death. The cell death occurred by the typical apoptotic cascade, namely DNA fragmentation, chromatin hypercondensation and margination, karyorrhexis, ribonucleoproteins segregation and cytoplasm cleavage; in addition some necrotic cells were found. The data indicates that the hyperthermic treatments limit the cell proliferation (e.g. arrest and/or deceleration of the cell cycle) by facilitating the trigger of programmed cell death. It was concluded that thermal injury can be considered an effective inducer of antiproliferative and apoptogenic associated effects on the growth of this kind of neoplasia.

Hyperthermia-induced cell death by apoptosis in myeloma cells.

We have analyzed by morphological (TEM) and histochemical (TUNEL reaction) criteria the type of cell death occurring in one case of human multiple myeloma after hyperthermia. Samples of cells examined immediately at the end of two treatments with a 15-day interval showed a significant degeneration, mostly demonstrating features of apoptosis (cell shrinkage, DNA fragmentation, karyorrhexis). The possible causes of the lag period between heating and apoptosis onset-expression are discussed.

Data of pre-clinical and early clinical trials of acriflavine and hydroxy-methyl-ellipticine reviewed, enriched by the experience of their use for 18 months to 6 years in combinations with other HIV1 virostatics.

Two virostatics which we discovered in 1990, acriflavine (ACF) and hydroxy-methyl-ellipticine (HEL) and shown active on HIV1 resistant to AZT have been introduced into combinations of four virostatics selected among ten available: themselves, plus zidovudine, zalcitabine, didanosine, lamivudine, stavudine, saquinavir, ritonavir, indinavir, the combinations were applied in 3-week sequences, differing from each other by drug rotation. Those which contained ACF may have more often a CD34 decrease than those containing neither ACF nor HEL, and they present more often a CD4 increase. No significant difference as far as side effects or beneficial effects could be detected after 18 months to 6 years, between sequences containing ACF or HEL or both, and sequences not containing any one of them.

Long-term primary human tumor cell cultures and mixed autologous tumor-lymphocyte cultures for adoptive specific antitumoral immunotherapy.

Anticancer immunotherapy may improve the body's cancer defences, quality of life and survival. Encouraging treatment models have been proposed, namely the non-specific LAK and ALT cell therapies; the relatively specific TIL/CTL cell therapy as well as allogenic cancer vaccines; autologous cancer vaccines. A procedure to prepare an autologous antitumor serum for cancer therapy using ex vivo activated autologous cells is here reported. Peripheral blood lymphocytes obtained by leukaphoresis were cultured with autologous tumor cells in RPM1 1640 and rh-IL-2 for 48 hours at 37 degrees C in a humidified, CO2 enriched atmosphere. The cytokine rich supernatant, as well as the activated cells in medium were frozen, lysed and aliquotted, frozen, and stored at -20 degrees C. The material was used for specific anticancer immunotherapy. Fibroblast-free tumor cells are being maintained in long term culture for future ex vivo sensitization of autologous cells.

Cytokine patterns in adults with AIDS.

BACKGROUND: It has been reported that in HIV infected patients enhanced production of IL-4 and IL-10 in response to stimulation of peripheral blood lymphocytes with phytohemagglutinin is associated with disease progression. Some have proposed that a switch from a cytokine profile associated with CD4+ Th1 predominance (IL-2, IFN-G, TNF-B) to Th2 predominance (IL-4, IL-5) plays a major role in the progression of HIV infection. Others find no clear evidence for the dichotomy of Th1 and Th2 predominance in HIV infected patients Discrepant results have been reported in studied populations in which only a few cytokines have been examined. METHODS: Thirty-one adult patients with AIDS but without other active infections provided serum and peripheral blood lymphocytes for determination of cytokine levels. Their responses were compared to those of five normal healthy volunteers without active infection. ELISA techniques were employed to quantitate cytokine levels. Both circulating lymphocyte and enriched lymphocyte populations were studied with and without stimulation employing phytohemagglutinin and/or rhIL-2. RESULTS: Patterns of cytokine expression were analyzed in 31 adult patients with AIDS but without other active infections. All had CD4+ cell counts below 50 and large viral loads (Roche PCR). The unstimulated cytokine profile in these patients generally showed marked elevations in IL-1, IL-3, IL-4, IFN-G, TNF-A, TNF-B, OSM, and TGF-B. Minimal elevations compared to normal healthy volunteers were noted for IL-2, IL-6, IL-8, IL-12, IFN-A, and IL-6SR. The levels of RANTES were lower than in normal healthy volunteers. Responses of peripheral blood lymphocytes to stimulation with phytohemagglutinin showed enhancement of all cytokines in all subjects studied though the response was much less marked in AIDS patients than in normal volunteers with the exception of IL-3, IL-4, IL-8, TNF-B, and TGF-B which are increased. Little difference in IL-2 and IL-12 expression was noted between stimulated peripheral blood lymphocytes of AIDS patients and normal healthy volunteers. No relation was noted with patient age or with any use of antiretroviral agents. Recombinant human IL-2 was a less potent stimulant than was phytohemagglutinin. CONCLUSION: The character of cytokine response in AIDS patients may be directly related to the stimulus employed in test systems. There is no evidence for Th1/Th2 dysregulation. Cytokine elevations in AIDS patients generally are reflective of chronic infection (the virus). Lymphocytes from AIDS patients do not respond as well to stimulation as do those from normal healthy volunteers. The stimulated lymphocyte response in AIDS patients suggests there is underlying low-grade host versus virus reaction in these patients (exaggerated responses of IL-3, IL-4, IL-8, TGF-B).

Hyperthermic effects on the human metastatic liver: a TEM study.

Using ultrastructural analysis, we studied the effects of hyperthermic treatment of one case of human liver metastasis from colon carcinoma. The results indicate that the main hyperthermic response involves the neoplastic and the histiocytic cell population. The drastic decrease in metastatic cells was accompanied by the appearance of cell fragments and apoptotic bodies. Consequently, the histiocytic component (Kupffer cells) showed increased frequency, indicating an activated state. The data are consistent with a direct action of the heat on tumor cells with subsequent activation of Kupffer cells.

Higher survival of refractory metastatizing breast cancer after thermotherapy and autologous specific antitumoral immunotherapy.

46 women (average 54.3 yrs) with refractory metastatizing breast cancer were treated with thermotherapy and autologous specific immunotherapy (rhIL-2 ex vivo activated cells). Metastases involved one organ in 69%; in particular, bone, lung, liver. The PS after treatment was satisfactory in 41%; the outcome was better in those cases with metastases to one site. The women remaining alive were 31/46; 67% of thermoimmunotherapy treated patients were alive after a maximum survival time of 85 months (median 24 months). The 36 months of control showed a 5-fold higher survival rate in our series when challenged with that of compared women undergoing only chemotherapy (p < .001).

Lysosomal exocytosis induced by hyperthermia: a new model of cancer death. III. Effect on liver metastasis.

The purpose of the present study was to evaluate by morphological approaches (light and electron microscopy), the effect of hyperthermic treatment in one case of human liver metastasis. The results demonstrate that hyperthermia causes a significant reduction of the metastatic cells circulating into sinusoids and the "normalization" of the hepatocytes substructure. The data are consistent with a direct and/or indirect action of the temperature on the presence of infiltrating tumor cells. Particular importance is attributed to a general activation of lysosomes present in neoplastic cells, Kupffer cells and hepatocytes.

AIDS therapy with two, three or four agent combinations, applied in short sequences, differing from each other by drug rotation. I. First of two parts: a phase I trial equivalent, concerning five virostatics: AZT, ddI, ddC, acriflavine and an ellipticine analogue.

We have individually treated ten AIDS patients whose CD4 numbers were inferior to 200/mm3, with the five following HIV1 virostatics: a) azido-deoxythymidine (AZT), dideoxyinosine (ddI) and dideoxycytidine (ddC), which affect the same viral target, retrotranscriptase, b) acriflavine (ACF) and methyl-hydroxy-ellipticine (MHE) which we have discovered to be strong virostatics in vivo, in mice, against Friend's virus, and in man, against AZT resistant HIV1. We have shown that their combinations with AZT, hitting three viral targets, reduces in mice, the blood Friend's virus load below detectable level. Due to the short doubling time of HIV1, AIDS therapy must be continuous, and to allow the best tolerance, the five virostatic combinations were applied in short, three-week sequences, each differing as much as possible from the former and from the following one, due to drug rotation [1]. Among the ten patients, a) three received the two-drug combinations for 15 to 30 months, followed by the three-drug combinations, b) three received the three-drug combinations from the beginning, c) four received the four-drug combinations also from the beginning, two having less than 10 CD4/mm3 at initiation of treatment, and two having more than 100. The tolerance was remarkable: the only side-effect being macrocytosis. The application of the two-drug combination sequences maintained stable CD4 levels in two subjects whose viral load (the evaluation of which had became available) was, at the end of this period, of 4,486 and 39,238 RNA copies. The third subject who had received, an intensive UV irradiation for a psoriasis, presented an irreversible decrease in his CD4 count and a high viral load (1,352,495 RNA copies/mL) at the end of the two-drug period. Fifteen to 25 months after the shift to the three-drug combinations, the viral load decreased, from 39,328 to 13,291 in one of the non-UV irradiated subjects, and from 1,352,495 to 314,387 in the irradiated one. No subject had an increase in CD4 number. In the three patients having initially received the three-drug combinations, a very strong decrease of viral load was registered after periods of observation varying from 77 to 40 months, while the CD4 counts increased moderately in two subjects, and noticeably in the third (from 126 to 266). Out of the four subjects initially treated with four-drug combinations, the two with less than 10 CD4/mm3 had a moderate decrease in viral load in about three months, and the CD4 increased from 9 to 34/mm3 in one. But the two subjects, because of opportunistic infections and psychological reasons, abandoned their treatments. In the two subjects who had more than 100 CD4/mm2 at initiation of the four-drug combination treatment, the viral load decreased to undetectable levels after four months: but their CD4 counts, after some oscillations, had very moderately increased at the end of the observation period (respectively, from 200 to 222, and from 129 to 134). In practice, these results suggest the interest of conducting phase II or III studies of AIDS treatment protocols, starting with the four-drug combination model, and attempting to maintain the effect with the three-drug combination one. As for theoretical considerations, one must underline the contrast between the remarkable reduction of the viral load and the usually moderate increase of the CD4 counts. The study but not the trial has been interrupted, due to the unavailability of three antiproteases, saquinavir, ritonavir and indinavir, which are now introduced in the same type of combinations, one by one, in replacement of one of the studied agents as shown in figure 1. The effect of increasing the total number of virostatics from five to eight will be published in the second part of this article series.

Therapeutic hyperthermia in cancer and AIDS: an updated survey.

The aim of this paper is to update with personal contributions the progress thus far accomplished in the clinical application of hyperthermia (HT) in cancer and chronic infectious diseases. The HT treatment has been successfully developed since the 1970s in cancer patients in whom it showed positive results consisting of complete or partial clinical remissions. Its rationale was based on the fact that core temperatures of > or = 42 degrees C induce cytotoxic effects that are higher in malignant cells than in normal cells. HT could be applied by different methods according to type, stage, and localization of the malignancies. Thus, systemic whole-body HT (WBH), through invasive or noninvasive techniques, was first used in disseminated cancers; local perfusion, infusion, and interstitial HTs have been applied in limb, skin, subcutaneous, or intracavitary tumors. The observation of a macrophagic lysosomal exocytosis and subsequent cancer cell death induced by HT, suggested that its mechanism of action involves an immune reaction. This suggested the possibility of associating HT with cytotoxic agents, antibiotics, antiviral drugs, and antioxidants, including beta-carotene (BC). The association of HT with BC at high doses are synergistic in patients with AIDS-related complex (ARC) and improve its symptoms, preventing the progress of the disease into the severe stage of AIDS; the same synergism helped also to increase the survival time in patients with severe AIDS.

Hyperthermia in the treatment of brain metastases from lung cancer. Experience on 17 cases.

Medium or long-term survival in metastatic "non oat cell" lung tumors is seldom possible only if surgery can eradicate the lesion. Out of 17 patients treated with hyperthermia plus nitrosoureas 16 (94%) responded, with clinical improvement, radiological regression or disease stabilization. The survival time of the improved patients was 12.7 months. Hyperthermia in combination with nitrosoureas seems to allow clinically and radiologically satisfactory responses in lung tumors metastatic to the brain.

Is metastatic breast cancer, refractory to usual therapy, curable?

Breast cancer with metastatic disease is presently incurable. Significantly shorter survival rates are seen in premenopausal women despite usual therapies when compared to survival rates in older women. Median survival rates of 24-30 months are documented in large-scale prospective clinical trials of previously untreated women with metastatic breast cancer regardless of the protocol employed (chemotherapy, hormone therapy). High dose chemotherapy followed by autologous stem cell rescue (bone marrow or peripheral blood) is associated with significant response and possibly improved survival in chemosensitive patients with metastatic disease without visceral metastases though with significant toxicities and cost (median survival rate of 20 months). Patients with refractory disease have dismal results regardless of therapy (median survival rates of 8-9 months in a number of prospective trials with or without stem cell rescue). The use of alpha-interferon in such patients has not improved response. Whole body hyperthermia is of benefit in the presence of liver metastases although median survival rate is in the range of 12 months. New treatment approaches with curative intent are clearly required. We report fifty-nine patients with metastatic breast cancer refractory to common therapies who were treated with whole body hyperthermia (40 degrees C) with low dose chemotherapy and immunomodulation: five presented with brain metastases; 13 with multiple bone metastases; 8 with liver metastases; 10 with lung metastases; and 23 with multiple soft tissue metastases. Fifteen were premenopausal; 44 postmenopausal. Twenty-three achieved complete remission. Fourteen have been sustained with patients remaining alive from 17-80 months (median, 40 months). Nine failed after 20-40 months of being disease-free (mean, 32 months).(ABSTRACT TRUNCATED AT 250 WORDS)

Whole body hyperthermia associated with beta-carotene supplementation in patients with AIDS.

The objective of this work was to check possible additive beneficial effects of whole body hyperthermia (WBH) associated with beta-carotene (BC) supplementation in patients with AIDS. In a pilot study, 10 HIV positive patients, (8 with AIDS and 2 with AIDS related complex, ARC), after AZT or DDI discontinuation, were first treated with one single session of WBH applied with a non-invasive procedure at 42 degrees C core temperature for one hour, and subsequently supplemented with BC 120 mg daily continuously. All patients well tolerated the non-invasive WBH as well as the high dose BC supplementation. Apart from one patient who died after 4 months, all the others underwent an HIV burden diminution, clinical improvement and amelioration of laboratory data, along with an subjective improvement of their life quality. With reference to control groups, namely (a) only WBH applied with extracorporeal procedure to 31 AIDS patients, and (b) only BC supplementation at high dosage applied to 64 ARC patients, the combined physical and BC supplemental treatments clearly showed a better and longer lasting response.

Lysosomal exocytosis induced by hyperthermia: a new model of cancer cell death. II. Effect on peritoneal macrophages.

Recently we described the "macrophagic lysosomal exocytosis" (MLE) as a possible new mode of cancer cell death induced by hyperthermia (HT) [1]. In order to confirm this first report, HT was applied at the peritoneum level with perfusional procedure, after catheter insertion under local anesthesia. We evaluated the subcellular changes of peritoneal macrophages in human gastric tumor, before and after hyperthermic treatment at 42 degrees C for 90 minutes. Transmission electron microscopy showed that treatment produced the disappearance of the cytoplasmic granules, with a consequent extracellular scavenger action of the phagocytic cells, the proliferation of some organelles such as mitochondria, endoplasmic reticulum and Golgi complex that may be addressed to a subsequent restoration of the granular pool in the degranulated macrophages. This phenomenon can enhance the already documented effect of hyperthermic perfusional chemotherapy in peritoneal tumors.

Systemic hyperthermia in the treatment of HIV-related disseminated Kaposi's sarcoma. Long-term follow-up of patients treated with low-flow extracorporeal perfusion hyperthermia.

Treatment of HIV and related malignancies with pharmacologic and biologic agents has not appreciably modified the course of disease. Immunologic impairment remains the critical factor in response. We report the long-term results of a single session of low-flow (0.3 L/min) extracorporeal perfusion hyperthermia on 29 men and 2 women with disseminated Kaposi's sarcoma and profound immunologic impairment. Any antiretroviral drug employed by the patient was stopped 72 hours prior to treatment and withheld during the period of follow-up. Core temperature was raised to 42 degrees C and held for 1 hour with extracorporeal perfusion and ex vivo blood heating to 49 degrees C as the means of temperature control. Of 31 patients, 2 died of complications secondary to treatment (cardiac arrhythmia; CNS bleed). There were two cases of intravascular coagulopathy. Pressure point skin damage may occur despite adequate cushioning. At 30 days posttreatment complete or partial regressions were seen in 20/29 of those treated, with regressions persisting in 14/29 of those treated by 120 days posttreatment. At 360 days, 4/29 maintain tumor regressions with 1 in complete remission (at 26 months). The patient in complete remission remains culture-negative and PCR-negative for HIV. CD4+ counts rose from around 250 to, and remain around, 800 in this man. Selected healed lesions were biopsied to demonstrate tumor absence. Patients were selected for treatment if pretreatment testing of the tumor showed regression in vitro with heat exposure. Analysis of the early and midterm failures showed little sustained rise of the CD4+ cells if presenting total CD4+ counts were below 50 and had been at such low levels for extended periods, although other surrogate markers of HIV activity declined (semiquantitative PCR) during this period and is felt to support the hypothesis of apoptosis proposed in this illness. Analysis of the tumors of the few men not responding demonstrated elevated levels of IL-6 as compared to responders (12 vs < 1 pg/ml). At 120 days 29/31 patients remained alive (expected, 20). At 360 days, 21/31 remained alive (expected, 11). In no patient was HIV activity stimulated with heat exposure. CMV retinitis did clear in some patients treated (both techniques), but treatment alone did not prevent later development of retinopathy. EBV parameters were markedly altered in the short term with heat exposure in some patients. Few patients showed herpes simplex activation. Varicella-zoster virus remitted in some patients. There is utility in the use of systemic hyperthermia to control HIV and related malignancy.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo eradication of Friend virus as an experimental HIV-model, by combination of zidovudine, acriflavine and an ellipticine analogue. Possible application to the treatment of human pre-AIDS?

Simultaneous administration of zidovudine, acriflavine and celliptium to Friend virus-injected mice eradicates the virus, as evidenced by the impossibility of the treated-mouse serum, when injected to virgin recipients, to induce spleen foci formation. An adapted preliminary protocol given to patients in whose p 24 antigen was present in the blood, lead to a considerable reduction of that marker. The cures lasted 3 weeks, and were repeated after 3-week intervals. Since p 24 antigen returns to pre-treatment levels at the end of the interval, research should concentrate on the maintenance of the effect during the interval.

Tumor angiogenesis: evidence of new blood channels from plasma infiltrations.

Stroma formation in Ehrlich carcinoma, studied with histochemical and TEM techniques, is similar to wound healing. In this tumour mast cells, macrophages, adipocytes, platelets and fibrin seem to co-operate locally with malignant cells in regulating stroma formation. The gaps opened in the tumor parenchyma by plasma outpouring from local blood vessels seem to offer easy routes for endothelial cell migration towards ill-nourished areas, and may explain the irregular aspect of tumor microvascularity.