Ultrastructure of intraerythrocytic Babesia microti with emphasis on the feeding mechanism

Babesia microti is a highly polymorphic organism. To unravel its fine structure and the function of organelles it was necessary to resort often to serial sections. A single plasma membrane covers the organism. In trophozoites approaching reproduction, segments of double membranes can be found below the plasma membrane. In electron micrographs of poor resolution these segments of double membranes look like pieces of thick membranes and they were often thought to be a thick 2nd membrane. Before the segments of double membranes appear 2 other organelles are formed in older trophozoites: micronemes and rhoptries. There are indications that these structures originate from vesicles of the Golgi apparatus. Large dense bodies of the same structure as the host cytoplasm are not food vacuoles but merely invaginations of host cytoplasm, as found in serial sections and in organisms removed from the host cell. Feeding in Babesia seems to take place by a special organelle composed of tightly coiled double membranes located partly inside and partly outside the parasite. It is assumed that extracellular digestion of host cytoplasm take place through this organelle. The nucleus remains undifferentiated throughout the whole intraerythrocytic stage. It becomes irregular, loboid, but does not divide and remains a single body until the late stage of reproduction when only a small portion, a bud, extends into the forming merozoite.     

Cosegregation of a novel Bartonella species with Borrelia burgdorferi and Babesia microti in Peromyscus leucopus

The present study was designed to investigate the effect of prolactin (PRL) on plasminogen activator inhibitor-I (PAI-I) and tissue type plasminogen activator (tPA) gene expression in eCG-primed granulosa cells in vitro. At 46 h after the hormone treatment, ovaries were removed, and granulosa cells were prepared for culture. Cells were incubated for various times in serum-free medium in the presence or absence of LH and PRL alone or in combination. tPA and PAI-I activities in the media were assayed by fibrin overlay and reverse fibrin autograph, respectively. Cytoplasmic RNA from granulosa cells was prepared using the NP-40 method and was assayed for PAI-I and tPA mRNA levels. We demonstrated the following. 1) PRL increased PAI-I mRNA production in cultured granulosa cells. Inclusion of LH with PRL had a synergistic effect on increasing PAI-I mRNA levels. After 48-h culture, 3-fold increases in PAI-I mRNA levels were seen with LH in combination with PRL as compared with PRL alone. The synergistic increase in PAI-I mRNA levels occurred in a dose- and time-dependent manner. 2) The increase in PAI-I mRNA synthesis by PRL alone, or by PRL in combination with LH, was well correlated with the changes in PAI-I activity and antigen levels in the conditioned media. 3) PRL in the culture also dramatically decreased LH-induced tPA mRNA and activity in a dose- and time-dependent fashion. The decrease in the tPA activity by PRL was also correlated with an increase in the amount of PA-PAI-I complexes in the cell-conditioned media. 4) In situ hybridization of tPA and PAI-I mRNAs in the cultured granulosa cells also showed that PRL was capable of enhancing PAI-I mRNA while diminishing tPA mRNA production induced by LH. This suggests that the dose- and time-dependent decrease in the gonadotropin-induced tPA activity in the culture by the presence of PRL may be due to decreasing tPA mRNA synthesis on one hand and to neutralization of the tPA activity by the increased PAI-I activity on the other.     

Comparative pathogenesis of human WA1 and Babesia microti isolates in a Syrian hamster model

The medial thigh flap is a perforator-based flap nourished with septocutaneous or muscle perforators originating from the femoral vessels. To date, 8 patients have been repaired with this flap and extended or connected flaps including this flap: 4 patients with lower leg defects and 4 patients with intraoral and neck defects. The advantages of this flap are (1) several pedicle perforators exist for this flap, which makes possible duplicated vascular anastomoses to establish reliable circulation of the transferred flap; (2) the flap can be extended or connected to other neighboring flaps in the anterior thigh, so that extensively wide defects can be closed in one stage; (3) the great saphenous vein can be simultaneously used as a vein graft or for venous drainage for the flap; (4) the anterior branch of the femoral nerve can be used for sensory potential; and (5) there is minimum morbidity of the donor defect and a large dominant vessel for the leg can be preserved. The suitable indications for this flap are defects after removal of skin cancer in the foot or lower leg and wide defects after resection of head and neck cancer, which can be reconstructed with the flap connected to neighboring skin flaps. The disadvantages of this flap are that it has a small, short vascular pedicle and the bulkiness of the flap's fatty tissue often requires thinning.     

Different peptide binding specificities of hsp70 family members

A set of heptapeptides was used to compare the relative peptide affinities of three proteins of the hsp70 family: bacterial DnaK, mammalian cytosolic hsc70, and BiP from mammalian ER. Each hsp displays a characteristic pattern of relative affinities. DnaK and hsc70 are more similar to each other than to BiP. A difference in peptide binding specificity may be an important determinant in adjusting an hsp70 family member to its particular cellular function.     

Effect of herbimycin A on hsp30 and hsp70 heat shock protein gene expression in Xenopus cultured cells

Glucose intolerance and diabetes mellitus are both prevalent in patients with chronic liver diseases. We examined the efficacy and systemic safety of therapy with an alpha-glucosidase inhibitor, acarbose, in diabetes mellitus associated with chronic liver diseases. Twenty patients with chronic hepatitis or liver cirrhosis and overt diabetes mellitus received acarbose (taken orally) for 8 weeks. The initial dosage of acarbose was 50 mg three times daily, taken before meals; this was increased to 100 mg three times daily after 2 weeks. The mean fasting plasma glucose level was 173.7 +/- 18.6 mg/dl (mean +/- SE) at entry, and was significantly decreased to 132.9 +/- 7.5 mg/dl (P < 0.05) after 8 weeks of acarbose treatment. The improved glycemic control was reflected by a significant decrease in glycosylated hemoglobin (HbA1c) from 7.2 +/- 0.3% at entry to 6.3 +/- 0.2% (P < 0.05) after 8 weeks. Serum levels of both aspartate and alanine aminotransferases fluctuated during acarbose treatment, probably due to the natural course of chronic liver diseases, but the mean values had decreased after 8 weeks of treatment. Plasma ammonia levels increased, from 61.3 +/- 10.7 micrograms/dl to 71.1 +/- 9.6 micrograms/dl after 8 weeks of acarbose treatment but the increase was not significant. Clinically significant elevation of plasma ammonia concentration was seen in 2 cirrhotic patients (121 and 124 micrograms/dl); this was asymptomatic and gradually returned to the normal range despite continuous acarbose treatment in one patient, and was reversed after the withdrawal of acarbose with the concomitant administration of lactulose in the other patient. No other blood tests results, including albumin, cholinesterase, and prothrombin time, or lipid profile and nutritional status, in terms of rapid turnover proteins, prealbumin, retinol binding protein, and transferin, were altered throughout the study period. These results indicate that diabetes mellitus associated with chronic liver diseases may be safely and effectively treated with acarbose. However, clinicians must be aware of the possibility of hyperammonemia when they prescribe acarbose for patients with diabetes mellitus and advanced liver cirrhosis.     

The role of DnaJ-like proteins in glucocorticoid receptor.hsp90 heterocomplex assembly by the reconstituted hsp90.p60.hsp70 foldosome complex

The glucocorticoid receptor (GR) is recovered from hormone-free cells in a heterocomplex with the molecular chaperone hsp90, which is required to produce the proper folding state for steroid binding. GR.hsp90 heterocomplexes are formed by a multiprotein system that appears to exist in all eukaryotic cells. Recently, we have reconstituted a receptor.hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60. We have shown that hsp90, p60, and hsp70 form an hsp90.p60. hsp70 complex that converts the GR from a non-steroid binding to a steroid binding form (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). The resulting GR.hsp90 heterocomplex rapidly disassembles unless p23 is present to bind to the ATP-dependent conformation of hsp90 and stabilize its association with the receptor (Dittmar, K. D., Demady, D. R., Stancato, L. F., Krishna, P., and Pratt, W. B. (1997) J. Biol. Chem. 272, 21213-21220). In the current work, we show that the purified rabbit hsp70 utilized in prior studies is contaminated with a small amount of the rabbit DnaJ homolog hsp40. Elimination of the hsp40 from the purified GR.hsp90 assembly system reduces assembly activity, and the activity is restored by addition of the purified yeast DnaJ homolog YDJ-1. hsp40 is a component of the hsp90.p60.hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60. Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, molybdate), a five-membered (p23. hsp90.p60.hsp70.hsp40) complex of chaperone proteins is formed in reticulocyte lysate or from purified proteins. The hsp40-free, purified assembly system has a modest level of assembly activity that is maximally potentiated by YDJ-1 when it is present at about one-twentieth the concentration of hsp70. Although hsp40 is not in the final GR.hsp90 heterocomplex isolated from L cell cytosol, it is in the GR.hsp90 heterocomplex assembled in reticulocyte lysate. We conclude that hsp40 is a component of the multiprotein hsp90-based chaperone system where it potentiates GR.hsp90 heterocomplex assembly.     

Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery

Hop, an abundant and conserved protein of unresolved function, binds concomitantly with heat shock protein 70 (Hsp70) and Hsp90, participates with heat shock proteins at an intermediate stage of progesterone receptor assembly, and is required for efficient assembly of mature receptor complexes in vitro. A largely untested hypothesis is that Hop functions as an adaptor that targets Hsp90- to Hsp70-substrate complexes; if true, then loss of either Hsp70 binding or Hsp90 binding by Hop should equally disrupt its ability to promote assembly of mature receptor complexes. To generate Hop mutants that selectively disrupt heat shock protein interactions, highly conserved amino acids in the previously mapped Hsp70 and Hsp90 binding domains of Hop and in a conserved C-terminal domain were targeted for small substitutions and deletions. In co-precipitation assays, these mutants displayed selective loss of association with heat shock proteins. In assays using Hop-depleted rabbit reticulocyte lysate for the cell-free assembly of receptor complexes, none of the Hop mutants inhibited Hsp70 binding to receptor, but all mutants were defective in supporting Hsp90-receptor interactions. Thus, Hop has a novel role in the chaperone machinery as an adaptor that can integrate Hsp70 and Hsp90 interactions.     

Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated

OBJECTIVES: We sought to evaluate the immediate angiographic results and intermediate-term follow-up after percutaneous treatment of left main coronary stenoses in the new device era. BACKGROUND: Historically, balloon angioplasty of left main coronary stenoses has been associated with high procedural morbidity and poor long-term results. It is not clear whether new devices are more effective in this anatomic setting. METHODS: Between July 1993 and July 1995, we performed initial left main coronary interventions on 46 patients (mean age 67 +/- 12 years, 26% women). Quantitative angiography was available for 42 of 46 interventions, and clinical follow-up was obtained for all patients at 1 month, 6 months and 1 year after initial revascularization. RESULTS: Most interventions (42 of 46) were performed in patients with "protected" coronary stenoses to the left coronary system owing to the presence of one or more patent left main coronary grafts. Seventy-seven percent of screened patients were deemed unsuitable for repeat coronary artery bypass surgery. Procedures performed included stenting in 73% of patients (alone in 30% and after rotational atherectomy in 43%), rotational atherectomy in 58% (alone in 15% and before stenting in 43%), directional atherectomy in 4% and angioplasty alone in 7%. Initial procedural success was achieved in all interventions, with no deaths, myocardial infarctions (creatine kinase, MB fraction > 50 IU/liter) or emergent bypass surgery. Follow-up data to date (median duration 9 months, range 6 to 19) demonstrate a 98% overall survival rate and a 6-month event-free survival rate of 78% (six target vessel revascularizations [TVRs], four non-TVRs). CONCLUSIONS: Treatment of protected left main coronary artery stenoses can be accomplished safely and effectively with new device technology. Intermediate-term follow-up demonstrates an acceptably low rate of death, myocardial infarction or repeat revascularization at 6 months and 1 year.     

Exogenous heat shock protein hsp70 activates potassium channels in U937 cells

With the use of patch clamp technique, the effect of exogenous heat shock protein hsp70 on ion channel properties in the plasma membrane of human promonocyte U937 cells has been examined. Cell-attached experiments showed that the addition of 30-100 micrograms/ml hsp70 to the pipette solution resulted in an activation of outward currents through potassium-selective channels of 9 pS unitary conductance. The activity of K(+)-selective channels did not depend on membrane voltage and could be controlled by the intracellular free calcium concentration as revealed in inside-out recordings. K+ channels with similar conductance and kinetic behaviour were found in normal cell-attached patches very rarely. Outside-out experiments showed that the addition of hsp70 to the external solution induced a channel-like stepwise increase of inward current which may provide cation entry from the extracellular medium. The interaction of extracellular hsp70 with the membrane surface of the native cell and of the excised fragment was found to be different. The results suggest that hsp70-induced activation of Ca-dependent K channels in monocyte-macrophage cells may be due to a local increase of free Ca2+ concentration just near the inner membrane side.     

Subarachnoid hemorrhage induces c-fos, c-jun and hsp70 mRNA expression in rat brain

To detect stress responses of the brain to subarachnoid hemorrhage (SAH), we investigated the expression of immediate early genes (IEGs) and hsp70 mRNA by in situ hybridization. Experimental SAH was produced in 49 rats by endovascular penetration. We also monitored the intracranial pressure (ICP) changes. The genes c-fos and c-jun were induced in the cerebral cortex, hippocampus and dentate gyrus in the penetrated side. mRNA coding for hsp70 was induced in the cerebral cortex, hippocampus, thalamus, hypothalamus and caudoputamen in the penetrated side and extended to the contralateral hemisphere. IEGs in the cerebral cortex were completely blocked by MK-801 pretreatment, but hsp70 mRNA was not. This suggests that the expression of IEGs correlates with spreading depression. The IEGs and hsp70 expression may reflect the severity of SAH impact and relate to the mechanisms of symptomatic vasospasm.     

Human stress protein hsp70: overexpression in E coli, purification and characterization

The gene encoding the stress-inducible member of human heat shock protein hsp70, was expressed in E. coli using the bacteriophage T7 RNA polymerase-based gene expression system. Recombinant hsp70 (R-hsp70) was purified from inclusion bodies after solubilization and refolding, using a combination of ATP-agarose affinity chromatography and ion-exchange chromatography. R-hsp70 was shown to be monomeric and free of its structurally similar E. coli counterpart, DnaK. In addition, R-hsp70 is functional as demonstrated by its ability to bind to peptides and to ATP. The availability of pure, correctly folded R-hsp70 in sufficient quantity will assist in the structural and functional characterization of hsp70. Furthermore, an understanding of the cytoprotective function of hsp70 and its role in immune responses during infections will be facilitated by the availability of pure R-hsp70.     

Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.     

Plant-derived amino acids increase hippocampal BDNF, NGF, c-fos and hsp70 mRNAs

Early alterations in mRNAs encoding neurotrophins and stress proteins were investigated following intracerebroventricular injections of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) in adult rats using in situ hybridization. Major increases in heat-shock protein 70, c-fos and brain-derived neurotrophic factor (BDNF) mRNAs were seen in hippocampus 1 h after BOAA or BMAA injections. Nerve growth factor mRNA was profoundly increased in the dentate gyrus (DG) after both treatments. Four hours after BMAA injections increased hybridization to BDNF mRNA was still seen in hippocampus, in parallel with reduced neurotrophin-3 expression in the DG. These alterations are in accordance with previous findings of BOAA and BMAA as potent glutamate receptor agonists.     

The different expression of proteins recognized by monoclonal anti-heat shock protein 70 (hsp70) antibody in human colonic diseases

Analysis of the expression of hsp70 (70 kDa heat-shock proteins) in normal and pathological tissues might prove their potential diagnostic and prognostic values. In the present study, we combined high-resolution two-dimensional polyacrylamide gel electrophoresis with immunoblotting to study hsp70 expression in normal, preneoplastic and neoplastic colonic mucosa. By monoclonal anti-hsp70 antibody, recognizing both the constitutive and inducible forms of hsp70, we have detected six charge isoforms localized in the pH 5.1-5.3 range and in the molecular mass range of 68-69 kDa in normal colonic mucosa. Immunostaining of hsp70 in polypous and malignant tissues revealed qualitative as well as quantitative changes in the expression of more acidic isoforms of hsp70 in comparison with normal tissue. Furthermore, the different basic isoforms of hsp70 were detected in chronically inflamed colonic mucosa from patients suffering from ulcerative colitis (UC) or Crohn's disease (CD). Besides the standard hsp70 protein pattern, additional proteins with molecular masses of about 39, 40, 74 and 75 kDa were variably immunostained in normal and pathological specimens. These observations suggest that hsp70 expression may be closely linked to disease etiology and/or pathophysiology.     

Folding of the glucocorticoid receptor by the heat shock protein (hsp) 90-based chaperone machinery. The role of p23 is to stabilize receptor.hsp90 heterocomplexes formed by hsp90.p60.hsp70

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor.hsp90 heterocomplexes. We have reconstituted a minimal receptor.hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833-12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR.hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). In this work, we show that addition of p23 to native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR.hsp90 heterocomplexes prepared with the minimal (hsp90.p60.hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR.hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90.p60.hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR.hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90.p60.hsp70 forms a GR.hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR.hsp90 heterocomplex to inactivation and disassembly.     

Inactivation of tumor suppressor p53 by mot-2, a hsp70 family member

The mortalin genes, mot-1 and mot-2, are hsp70 family members that were originally cloned from normal and immortal murine cells, respectively. Their proteins differ by only two amino acid residues but exhibit different subcellular localizations, arise from two distinct genes, and have contrasting biological activities. We report here that the two proteins also differ in their interactions with the tumor suppressor protein p53. The pancytosolic mot-1 protein in normal cells did not show colocalization with p53; in contrast, nonpancytosolic mot-2 and p53 overlapped significantly in immortal cells. Transfection of mot-2 but not mot-1 resulted in the repression of p53-mediated transactivation in p53-responsive reporter assays. Inactivation of p53 by mot-2 was supported by the down-regulation of p53-responsive genes p21(WAF-1) and mdm-2 in mot-2-transfected cells only. Furthermore, NIH 3T3 cells transfected with expression plasmid encoding green fluorescent protein-tagged mot-2 but not mot-1 showed an abrogation of nuclear translocation of wild-type p53. These results demonstrate a novel mechanism of p53 inactivation by mot-2 protein.     

Characterization of D10S and K71E mutants of human cytosolic hsp70

To determine the effect of mutations at the nucleotide-binding site of recombinant Hsp70 on its interaction with protein and peptide substrates, point mutations were made at D10 and K71, two residues at the active site. The D10S mutation weakened both ATP and ADP binding, while the K71E mutation weakened only ATP binding. In binding experiments using Hsp70 with no bound nucleotide, the mutated Hsp70s interacted with clathrin and peptide just like the wild-type Hsp70. However, the D10 mutation completely abolished the effects of both ATP and ADP on peptide and clathrin binding. The K71 mutation also abolished the effect of ATP on substrate binding, but ADP, which still bound tightly, had its normal effect on substrate binding. In addition, the D10S and K71E mutants had greatly reduced ability to uncoat clathrin-coated vesicles at pH 7.0, bind to clathrin baskets at pH 6.0, and undergo polymerization induced by YDJ1 in the presence of ATP. We conclude, first, that nucleotides must bind strongly to Hsp70 to affect substrate binding and, second, that interaction of Hsp70 with DnaJ homologues may also require a strongly bound ATP.     

A hsp70-2 mutation recognized by CTL on a human renal cell carcinoma

We performed T cell cloning experiments with a tumor-infiltrating lymphocyte subpopulation derived from a renal cell carcinoma tumor site (RCC-7) in which the TCR clonotypic repertoire had been analyzed in terms of TCRBV complementarity-determining region 3 size distribution. We report in this work the characterization of one of the five RCC-specific MHC class I-restricted CTL clones isolated in RCC-7. This TCRBV6J1S1 CTL recognized only the autologous RCC-7 tumor cell line in the context of HLA-A*0201, and the Ag is encoded by a mutated form of the hsp70-2 gene found in the tumor cells, but not in autologous PBLs nor in 47 other tumors. The identification of this gene was achieved by cotransfecting into COS cells a cDNA library of RCC-7 together with HLA-A*0201. Transfectants expressing the Ag were identified by their ability to stimulate TNF release by the CTL clone. The antigenic peptide is a decamer with a mutated residue at position 8. Half-maximal lysis was obtained with only 5 x 10(-11) M of decapeptide in target sensitization assays compared with 5 x 10(-8) M for the wild-type decapeptide. This difference in recognition was not related to difference in binding HLA-A*0201-presenting molecules, as assessed in an immunofluorescence-based peptide-binding assay using T2 cells. Constitutive hsp70 expression in various tumors suggests that this stress-induced protein may be recognized in situ by tumor-infiltrating lymphocytes. The finding in the tumor of a mutated form of the stress-induced hsp70-2 gene whose product is specifically recognized by TILs with high avidity is discussed in view of the present use of mycobacteria or heterologous heat-shock proteins as immunomodulators or as subunit vaccine candidates.