Effective anti-tumor immunity induced in mice by a two-step vaccination protocol.

BACKGROUND: TS/A cells (a Balb/c-derived tumor cell line), when injected into syngenic mice, give rise to rapidly growing tumors. In this study, a vaccination protocol was established which was able to elicit an immune response effective in controlling tumor growth. MATERIALS AND METHODS: T19.2.1, a TS/A clone enginereed to stably express the mycobacterial cell wall-associated 19-kDa lipoprotein, was used as cell vaccine to immunize Mycobacterium Bovis-BCG pre-immunized Balb/c mice. RESULTS: Mice receiving the two-step vaccination protocol were able to develop a strong anti-TS/A DTH reaction. Moreover, following a challenge with wild-type TS/A cells, some vaccinated animals rejected the tumor and the remaining animals showed a significantly increased survival in respect to controls. CONCLUSION: The expression on TS/A cells of the mycobacterial 19-kDa antigen, recognised in the context of a pre-existing memory immune response, promotes the immunological recognition of the otherwise non-immunogenic wild-type TS/A cells.

Anti-p53 and anti-heat shock proteins antibodies in patients with malignant or pre-malignant lesions of the oral cavity.

BACKGROUND: Evaluation of circulating anti-p53 antibodies is an easy-to-perform, widely employed, procedure to assess the p53 status in cancer patients. MATERIALS AND METHODS: Levels of circulating anti-p53 antibodies in patients affected either by oral SCC or by pre- malignant oral lesions were assayed using a commercial ELISA kit. Autoantibody titers to Hsp60 and Hsp72 were determined by conventional ELISA. RESULTS: Anti-p53 antibodies were detected in 3 out of 16 SCC-bearing patients (18.7%) and in 9 out of 13 patients suffering from pre-malignant oral lesions (69.2%). High titers of anti-Hsp60 autoantibodies were detected in 3 out of 29 patients (10.3%), while in all patients anti-Hsp72 titers were in the normal range. CONCLUSION: The presence of anti-p53 antibodies in both SCC-bearing patients and in patients with pre-malignant lesions support the notion that p53 gene mutation is an early event in oral tumorigenesis and suggest that this assay could be useful for diagnostic screening of pre-neoplastic lesions at high risk of recurrence and/or transition towards overt malignancy.

Different expression of CD44, ICAM-1, and HSP60 on primary tumor and metastases of a human pancreatic carcinoma growing in scid mice.

Surface adhesion molecules play an important, but still not completely clarified, role in tumor metastasization. In this research, FACS analysis was employed to analyze surface expression of CD44H, CD44v5, CD44v6, ICAM-1 and HSP60 in human pancreatic adenocarcinoma cells growing in vitro or collected ex vivo from primary tumors and lung metastases of tumor-engrafted SCID mice. It was found that, in metastatic cells, the standard form of CD44 (CD44H) is down,-regulated, while a large fraction of cells express on membrane the splice variants v5/v6 and, in addition, ICAM-1 and HSP60. It was also apparent that two cell populations are present in lung metastases: a CD44neg population, including cells expressing CD44v5/v6, ICAM-1 and HSP60 and a population of CD44pos, CD44v5/v6neg, ICAM-1neg and HSP60neg cells. These results demonstrate that, in pancreatic adenocarcinomas, metastasization is correlated with expression of the CD44 variants v5 and v6. Moreover, this is the first report demonstrating HSP60 surface expression on metastatic cells.

Induction and membrane expression of heat shock proteins in heat-treated HPC-4 cells is correlated with increased resistance to LAK-mediated lysis.

In human pancreatic carcinoma cells (HPC-4), a hyperthermic treatment at 43 degrees C for 30 min resulted in the vigorous induction of Hsp72, along with a less pronounced increase in the rate of synthesis of Hsp90, Hsp60 and Hsp 27. Biotinylation of surface-exposed proteins, followed by isolation of biotin-tagged proteins by affinity chromatography, demonstrated that both Hsp72 and Hsp60 are expressed on plasma membrane. Membrane expression of these two Hsps was confirmed by immunoprecipitation of surface biotinylated proteins with anti-Hsp72 and anti-Hsp60 specific antibodies. Cytotoxic assays showed that untreated HPC-4 cells are intrinsically resistant to NK-mediated lysis, while they were efficiently killed by LAK lymphocytes, as well as by exposure to TNFalpha. Following heat-treatment, cells became much more resistant to LAK-mediated lysis, while their sensitivity to NK-mediated lysis and to TNFalpha cytotoxicity remained unmodified.

Anorectal disease in HIV-infected patients.

PURPOSE: Anorectal diseases are common in human immunodeficiency virus-infected individuals. The aim of this prospective study was to assess the cause and clinical presentation of anorectal disease in this human immunodeficiency virus-infected population. METHODS: A registry of all human immunodeficiency virus-seropositive patients with anorectal complaints who were referred to and followed up in the colorectal surgery clinic at a county hospital was maintained, with all data collected prospectively. All patients underwent examination under anesthesia with random cultures and biopsies, along with specific sampling of any suspicious lesions. RESULTS: Data from 180 consecutive human immunodeficiency virus-seropositive patients with anorectal symptoms were analyzed. Mean age of the population was 34 years, with a male-to-female ratio of 14:1. This group comprised homosexual and bisexual males (55 percent), injection-drug users (15 percent), heterosexuals (12 percent), and others (18 percent). The average lag time from diagnosis of human immunodeficiency virus to anorectal symptoms was 48 months. The average CD4 lymphocyte count was 160 cells/mm3. The most common symptom was anorectal pain (57 percent), followed by lumps or warts (28 percent), rectal bleeding (12 percent), discharge (11 percent), and pruritus (6 percent), with 24 percent of patients having multiple complaints. Anal condyloma was the most prevalent pathology observed (43 percent), of which 10 percent was associated with anal intraepithelial neoplasia. Wide-based anal ulcers were the most frequent noncondylomatous lesions, occurring in 32 percent of patients, with the majority (91 percent) presenting with the chief complaint of anorectal pain. Some of these ulcers were associated with viral infections: herpes simplex virus (12 percent) and cytomegalovirus (7 percent). However, most ulcers were idiopathic, with negative cultures and biopsies. Other lesions encountered included fistulas (14 percent), abscesses (12 percent), hemorrhoids (6 percent), and malignancy, with two cases of Kaposi's sarcoma, one case of non-Hodgkin's lymphoma, and one case of squamous-cell carcinoma. More than one anorectal condition was identified in 16 percent of the patients. CONCLUSIONS: Human immunodeficiency virus infection is associated with a wide spectrum of anorectal disease, of which the most common lesions are anal condylomata and painful ulcers. The majority of these anal ulcers gave negative culture and biopsy results. In addition, there seems to be a high incidence of anorectal neoplasia in this patient population.

Cell surface localization of the 78 kD glucose regulated protein (GRP 78) induced by thapsigargin.

In the present study it was found that the synthesis of the 78 kD glucose-regulated protein (GRP 78 or BIP) is vigorously induced in human rabdomiosarcoma cells (TE 671/RD) following both short-term (1 h) and prolonged (18 h) exposure to 100 nM thapsigargin (Tg). Flow cytometric analysis with a specific anti-GRP 78 polyclonal antibody showed that Tg-treated cells express the GRP 78 on the plasma membrane. Cell surface localization of the Tg-induced GRP 78 was confirmed by biotinylation of membrane-exposed proteins and subsequent isolation of the biotin-labelled proteins by streptavidin/agarose affinity chromatography. It was found that a fraction of the Tg-induced GRP 78 is present among the biotin-labelled, surface-exposed, proteins. Conversely, the GRP 78 immunoprecipitated from unfractionated lysates of Tg-treated and biotin-reacted cells was found to be biotinylated. This is the first report demonstrating surface expression of GRP 78 in cells exposed to a specific GRP 78-inducing stimulus.

Anti-heat shock proteins autoantibodies in autoimmune thyroiditis. Preliminary study.

No data are currently available on the humoral autoimmune response against self heat shock proteins (hsps) possibly occurring in patients suffering from autoimmune thyroid disorders. Therefore, a preliminary research was carried out to assess whether anti-self hsps autoantibodies are present in human autoimmune thyroiditis (AIT). Serum samples from 14 patients with AIT were tested both in western blotting and in ELISA for antibody binding to protein extracts of human cultured cells (both unstimulated and heat-shocked) as well as to purified hsp 73 and hsp 72. It was found that 7 out 14 (50%) patients with AIT have autoantibodies of the IgM isotype strongly reacting with hsps 73/72. By contrast, autoantibodies to hsps 73/72 were detected only occasionally in sera from patients suffering from non-autoimmune thyroid disorders (1 out 11) as well as in sera from healthy individuals (2 out 9). No obvious clinical differences were detected between AIT patients who had autoantibodies to hsps 72/73 and those who did not. Clearly, more patients would have to be examined to determine whether the anomalous anti-hsps 73/72 autoimmune response occurring in a significant proportion of patients with AIT is in some way related to the pathogenesis and/or to the progression of the disease.

Characterization of a new high-temperature-induced 66-kDa heat-shock protein, antigenically related to heat-shock protein 72.

M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major "classic" heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45 degrees C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)+ RNA extracted from cells treated at 45 degrees C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)+ RNA extracted from cells heated at 42 degrees C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro. hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer.

Protein synthesis in TE 671/RD (human rabdomiosarcoma) cells treated with thapsigargin and hyperthermia: impairment of HSP 70 induction.

In this study we considered the quantitative and qualitative changes of protein synthetic activity occurring in TE 671/RD cells treated with thapsigargin (TG), with hyperthermia (HT) or with a combination of both these agents. In cells treated with TG (100 nM, continuous exposure), the overall protein synthetic activity was initially inhibited but subsequently recovered to about 60% of the initial level. Chronic TG exposure was also able to induce the expression of GRP 78. The rate of synthesis of GRP 78, after a lag period of about 2 h, increased gradually to reach a maximum (9-fold induction) after 6 h of TG-treatment and was then maintained at that level up to 18 h. A weak induction of GRP 94 was observed following 6-8 h of continuous exposure to TG. In cells treated with HT (43 degrees C for 30 min), a typical heat shock response was observed: in particular, the relative rate of synthesis of HSP 70 (the major heat-inducible mammalian heat shock protein) was increased 10-fold over the constitutive level. The heat-promoted induction of HSP 70 was significantly reduced by concomitant or previous exposure to TG. When TG and HT were administred simultaneously, the increase in HSP 70 synthesis was only 4.7-fold over the control level, while in cells pre-treated for 1 h with TG before the hyperthermic challenge the rate of HSP 70 synthesis was only stimulated 2-fold. In both these conditions, by contrast, it was apparent that HT did not affect the TG-promoted induction of GRP 78. The correlations between the TG-induced mobilization of cytosolic Ca2+ and the effects on protein synthesis are discussed.

Thermal behavior of a human glioma cell line and its response to combinations of hyperthermia and lonidamine.

The effect of hyperthermia and lonidamine, alone and in combination, on the clonogenic activity of a human glioma cell line was investigated. The time-temperature relationship of asynchronous, exponentially growing cells was defined in the range of 40-45 degrees C. All survival curves were exponential and an Arrhenius plot for heat killing was linear over the temperature range tested, with an activation energy of 192 Kcal/mol. The survival curve of lonidamine-treated cells was also exponential after an initial shoulder. The analysis of the interaction between lonidamine and hyperthermia, performed by the isobolar method, demonstrated an additivity of response so that the effectiveness of the combined treatment was the result of two independent effects. Lonidamine inhibits the neoplastic growth mainly through an ATP depletion, but the thermal killing was not mediated by the drug-induced changes in the energy status of the cell. The effectiveness of the combined treatment was strongly influenced by the schedule of administration. In fact, the sequence lonidamine-->hyperthermia made the cells less sensitive to heat so that the pre-established end-point, i.e. 30% survival, was never achieved whichever combination was used. This "drug-induced heat resistance" was not associated with the induction of heat shock proteins, but rather with modification of cell cycle. On the contrary, showing a purely additive effect, the sequence hyperthermia-->lonidamine allowed achievement of the pre-established cell killing (70%), with exposure times (1-2 hr) and with a temperature (42 degrees C) generally accepted as clinically achievable. Therefore, also considering its low systemic toxicity, lonidamine may be useful in reducing the side effects of hyperthermia.

Identification of a 66 kD heat shock protein (HSP) induced in M-14 human melanoma cells by severe hyperthermic treatment.

Following severe hyperthermic treatment M-14 cells synthesize at high rate a new protein of about 66 kD, in addition to the three well known major HSPs (HSP 28, HSP 70 and HSP 90). This 66 kD protein is constitutively expressed at low levels and its rate of synthesis is not enhanced by mild hyperthermic exposures (40 degrees C for 2-4 h; 42 degrees C for 1-3 h), sufficient to induce the three major HSPs. The 66 kD protein is induced whenever the thermal dose administered to cells attains a threshold, roughly corresponding to a 50% reduction in survival. The 66 kDa protein is not induced by a variety of compounds (disulfiram, arsenate, cadmium) able to elicit a stress response in M-14 cells, as indicated by enhanced synthesis of the three major HSPs. Once induced by a treatment at 45 degrees C for 15 min, the rate of synthesis of the 66 kD protein remains above the control level for 16-20 h during recovery from the stress, while the synthesis of HSP 70 is shut off between 8 and 12 h. Immunoblotting and immunoprecipitation studies showed that the 66 kD protein shares immunological determinant(s) with HSP 70. Pulse-chase experiments demonstrated that the 66 kD protein is not a degradation product or a late post-transcriptional modification of HSP 70. It is proposed that the 66 kD protein is a previously unrecognized heat shock protein (HSP 66), characterized by an unusually high threshold for its induction.

Survival response of a human glioma cell line to hyperthermia associated with rhein.

The effect of association of hyperthermia with the anti-inflammatory drug rhein (RH), 4,5-dihydroxyanthraquinone-2-carboxylic acid, on the clonogenic activity of human glioma cells has been examined. RH inhibits neoplastic growth mainly through an ATP depletion, but thermal cell killing is not mediated by the drug-induced changes in the energy status of the cell. The analysis of the interaction between RH and hyperthermia, performed with the isobolar method, demonstrates an additivity of the response so that the effectiveness of the combined treatment is the result of two independent effects. Although the effect of this combination is purely additive, RH allows us to achieve a pre-established cell killing with exposure times at 42 degrees C, which is generally accepted to be clinically achievable. RH might, therefore, be employed to reduce the side effects of hyperthermia without impairing its therapeutic effectiveness.

[Administration of lysine acetylsalicylate and meperidine in acute postoperative pain]. / Administración de acetilsalicilato de lisina y meperidina en el dolor agudo postoperatorio.

INTRODUCTION: Postoperative analgesia is insufficiently done due, among others, to the undesirable effects of analgesic agents. OBJECTIVE: The aim of this study was to analyze the effects of the simultaneous administration of opiates (meperidine) and AINES (lysine acetylsalicylate, ASL). MATERIAL AND METHODS: We studied 160 patients during the immediate postoperative phase. All of them underwent programmed surgery with the same general anesthetic technique. Patients were allocated into 8 groups of treatment: A) ASL 900 mg and 1.800 mg/8 h, B) ASL 900 mg and 3.600 mg/8 h, C) ASL 900 mg and meperidine 100 mg/8 h, D) ASL 900 mg and 1.800 mg/8 h together with meperidine 100 mg/8 h, E) meperidine 50 mg and ASL 1.800 mg/8 h, F) meperidine 50 mg and ASL 3.600 mg/8 h, G) meperidine 50 mg and 100 mg/8 h, and H) meperidine 50 mg and 100 mg/8 h together with ASL 1.800 mg/8 h. The effects of analgesic agents were evaluated on the basis of patient's appreciation of the degree of pain and relief and on the basis of an observer who did not know the therapeutic regime administered. Results were compared according to the analysis of variance in a graded factorial design. A p value less than 0.05 was considered significant. RESULTS: The degree of pain was significantly lower in groups C, D, G and H (specially in G and H) than in the remaining groups, but there were no significant differences between them. The lowest pain relief was observed in groups A, B, E and F. The highest attenuation of pain was achieved in groups G and H. The highest attenuation of pain was achieved in groups G and H. The observer considered that the two latter groups were those with the highest pain relief, followed by groups C and D. The remaining patients failed to show appreciable improvement. Nausea and vomiting only occurred in some patients after administration of a bolus of meperidine. There were no other secondary effects. CONCLUSIONS: The best degree of postoperative analgesia is achieved after administration of continuous infusion of meperidine 100 mg/8 h. Simultaneous infusion of ASL 1.800 mg/8 h did not improve the analgesia obtained with a bolus of 900 mg of ASL nor with a bolus of 50 mg of meperidine. Secondary effects were only nausea and vomiting and coincided with the administration of a bolus of meperidine.

Protein synthetic activity and adenylate energy charge in Rhein-treated cultured human glioma cells.

The effect of Rhein (RH) on the protein synthetic activity and adenylate energy charge in human glioma cells cultured in vitro has been investigated. The results demonstrate that in RH-treated cells, the protein synthesis is strongly decreased, but no modifications in the qualitative pattern occur. The extent of inhibition is a function of the drug concentration as well as of the time of exposure. Such an inhibition must be ascribed mainly to a reduction of adenylate energy charge brought about by RH because of its effect on respiration and glycolysis. The correlation between the adenylate energy charge and cell viability, as well as the possibility of using rhein as a biochemical modulator to reduce or to reverse multidrug resistance, are also discussed.

Stress response, survival and enhancement of heat sensitivity in a human melanoma cell line treated with L-canavanine.

L-Canavanine, like other aminoacid analogs, induces the synthesis of heat shock proteins (HPSs) but, unlike heat or other stressing agents, it fails to induce thermotolerance. We have studied the synthesis and the intracellular distribution of HSPs induced by canavanine, the effects of this analog on the viability and thermal sensitivity of a human melanoma cell line (M14) and the capacity of canavanine-induced HSPs to self regulate their own synthesis. Evidence indicates that the HSP induction is time--and dose--dependent and, also in the presence of arginine, is not associated with the development of thermotolerance. On the contrary, cells become more heat sensitive and are less efficient in the control of the feed-back mechanism that regulates HSP synthesis. The possible utilization of this substance as a potential aid for the treatment of tumors, in association with heat, was examined.

Preliminary evaluation of HER-2/neu oncogene and epidermal growth factor receptor expression in normal and neoplastic human ovaries.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In all the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.

Thermosensitization, heat shock protein synthesis and development of thermotolerance in M-14 human tumor cells subjected to step-down heating.

M-14 human tumor cells have been subjected to two regimens of step-down heating (SDH) consisting of a conditioning treatment at 42 degrees C for 1 h or at 44.5 degrees C for 20 min, immediately followed by heating at 40 degrees C. Both conditioning treatments thermosensitize the cells towards the subsequent heating at 40 degrees C; the thermosensitization ratio is 6.4 for cells conditioned at 42 degrees C for 1 h and 32.3 for cells conditioned at 44.5 degrees C for 20 min. The overall protein synthetic activity is reduced to 32.7% or 18.4% of control values following 1 h at 42 degrees C and 20 min at 44.5 degrees C, respectively; this inhibition is followed by a full recovery of the synthetic activity during the subsequent exposure at 40 degrees C. SDH-treated cells synthetize four heat shock proteins, with approximate molecular weights of 28, 64, 70 and 90 kDa. The pattern of HSPs induction observed in SDH-treated cells is similar to that found in cells subjected to single hyperthermic exposures. Cells subjected to the SDH sequence 42 degrees C/1 h-->40 degrees C/4 h develop thermotolerance, as indicated by a reduced sensitivity to further hyperthermic challenges.

Preliminary evaluation of HER-2/neu oncogene and epidermal growth factor receptor expression in normal and neoplastic human ovaries.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In all the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.

Inhibition of protein synthesis in neoplastic cells by rhein.

The action of rhein, 4,5-dihydroxyanthraquinone-2-carboxylic acid, on protein synthesis of neoplastic cells has been investigated. Rhein decreases amino acid incorporation in all cells tested. The inhibition of incorporation of labeled precursors into acid-insoluble material cannot be ascribed to an impairment of amino acid uptake, which is unaffected by the drug. Tests on cell-free system showed that rhein does not inhibit the TMV-mRNA directed in vitro protein synthesis, thus indicating that the protein machinery per se is not affected. The inhibition of protein brought about by the drug must be ascribed to an effect on the energy-yielding processes with a remarkable decrease in ATP content. The mechanism is similar to that of other metabolic inhibitors, but rhein, for its capability to inhibit both respiration and glycolysis, is effective at much lower concentrations.