Regression of engineered myeloma cells secreting interferon-gamma-inducing factor is mediated by both CD4(+)/CD8(+) T and natural killer cells.

IL-18 is a novel cytokine that stimulates T and NK cell activity and has potent antitumor effects. In this study, a mouse IL-18 gene was transfected into the mouse myeloma cell line J558. Our data demonstrated that (i) inoculation of 0.5x10(6) engineered tumor cells J558/IL-18 into syngeneic mice induced a Th1 dominant immune response and resulted in tumor regression in all 8/8 mice; (ii) the IL-18 antitumor effect was significantly decreased in mice depleted of either the CD4(+), or CD8(+), or NK cell subset, respectively but was completely abrogated in mice depleted of both CD4(+) and CD8(+) T cells; (iii) in vivo neutralization of IFN-gamma was accompanied by the growth of J558/IL-18 tumor in all the mice; and (iv) the J558/IL-18 tumor regression further induced protective immunity against a subsequent challenge by the parental J558 tumor, which is mediated by CD8(+) T cells as examined in the cytotoxicity assay in vitro and in the animal study in vivo. Taken together, our findings indicate that: (i) IL-18 can induce antitumor immune responses mediated by both CD4(+)/CD8(+) T cells and NK cells; and (ii) it is associated with IFN-gamma production. This study thus highlights the potential utility of IL-18 as an antitumor agent, a role that it can fulfil alone or in combination with other immunomodulatory cytokines such as IL-12.

Efficient antitumor immunity derived from maturation of dendritic cells that had phagocytosed apoptotic/necrotic tumor cells.

Dendritic cells (DCs) that acquired antigen from apoptotic tumor cells are able to induce major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes and antitumor immunity. In the present study, we investigated the efficiency of antitumor immunity derived from DCs that had phagocytosed apoptotic/necrotic BL6-10 melanoma cells compared with that of DCs pulsed with the tumor mTRP2 peptide. Our data showed that phagocytosis of apoptotic/necrotic tumor cells resulted in maturation of DCs with up-regulated expression of proinflammatory cytokines [interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, interferon-gamma and granulocyte-macrophage colony-stimulating factor], chemokines (MIP-1alpha, MIP-1beta and MIP-2), the CC chemokine receptor CCR7 and the cell surface molecules (MHC class II, CD11b, CD40 and CD86), and down-regulated expression of the CC chemokine receptors CCR2 and CCR5. These mature DCs displayed enhanced migration toward the CC chemokine MIP-3beta in a chemotaxis assay in vitro and to the regional lymph nodes in an animal model in vivo. Our data also showed that vaccination with DCs that had phagocytosed apoptotic/necrotic BL6-10 cells was able to (i) more strongly stimulate allogeneic T-cell proliferation in vitro, (ii) induce an in vivo Th1-type immune response leading to more efficient tumor-specific cytotoxic CD8(+) T-cell-mediated immunity and (iii) eradicate lung metastases in all 6 vaccinated mice compared with mice vaccinated with DCs pulsed with the tumor mTRP2 peptide, in which lung metastases were reduced (mean number of 16 per mouse) but not completely eradicated. Therefore, DCs that had phagocytosed apoptotic/necrotic tumor cells appear to offer new strategies in DC cancer vaccines.

Lymphotactin expression by engineered myeloma cells drives tumor regression: mediation by CD4+ and CD8+ T cells and neutrophils expressing XCR1 receptor.

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4(+) and CD8(+) T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.

Expression of transforming growth factor beta(1), beta(3), and basic fibroblast growth factor in full-thickness skin wounds of equine limbs and thorax.

OBJECTIVE: To map the expression of transforming growth factor (TGF)-beta(1), TGF-beta(3), and basic fibroblast growth factor (bFGF) in full-thickness skin wounds of the horse. To determine whether their expression differs between limbs and thorax, to understand the pathogenesis of exuberant granulation tissue. STUDY DESIGN: Six wounds were created on one lateral metacarpal area and one midthoracic area of each horse. Sequential wound biopsies allowed comparison of the temporal expression of growth factors between limb and thoracic wounds. ANIMALS: Four 2- to 4-year-old horses. METHODS: Wounds were assessed grossly and histologically at 12 and 24 hours, and 2, 5, 10, and 14 days postoperatively. ELISAs were used to measure the growth factor concentrations of homogenates of wound biopsies taken at the same timepoints. RESULTS: TGF-beta(1) peaked at 24 hours in both locations and returned to baseline in thoracic wounds by 14 days but remained elevated in limb wounds for the duration of the study. Expression kinetics of TGF-beta(3) differed from those of TGF-beta(1). TGF-beta(3) concentrations gradually increased over time, showing a trend toward an earlier and higher peak in thoracic compared with limb wounds. bFGF expression kinetics resembled those of TGF-beta(1), but no statistically significant differences existed between limb and thoracic wounds. CONCLUSIONS: Growth factor expression is up-regulated during normal equine wound repair. TGF-beta(1) and TGF-beta(3) show a reciprocal temporal regulation. Statistically significant differences exist between limb and thoracic wounds with respect to TGF-beta(1) expression. CLINICAL RELEVANCE: The persistence of TGF-beta(1) expression in leg wounds may be related to the development of exuberant granulation tissue in this location, because TGF-beta(1) is profibrotic.

Cancer gene therapy by adenovirus-mediated gene transfer.

Cancer arises as a direct result of genetic mutations. It therefore stands to reason that cancer should be well suited for the correction through gene therapy. Recent advances in the understanding of the molecular pathogenesis of cancer and the rapid development of recombinant DNA technology have made cancer gene therapy feasible in the clinical setting. The current efforts for cancer gene therapy mainly focus on immunogene therapy, chemogene therapy, restoration of tumor suppressor gene function, and oncolytic virus therapy. Central to all these therapies is the development of efficient vectors for gene delivery--this remains a work in progress. These vectors can be classified as viral and non-viral vectors. This paper will concentrate on viral vectors because of their practical advantages over non-viral vectors. Of the viral vectors, by far the most important are the human adenoviruses as is reflected by the enormous data and literature accumulated by studies relating to animal tumor models and clinical trials. In this review, we examine the recent progress in adenovirus-mediated cancer gene therapy with regard to cytokine gene, tumor suppressor gene, chemogene, and oncolytic adenovirus. We also discuss the current limitations of the adenoviral vector system and how they may be circumvented in future developments relating to targeted gene delivery.

Adenovirus-mediated p16INK4 gene transfer significantly suppresses human breast cancer growth.

The p16INK4 tumor suppressor gene encodes a protein that inhibits cyclin-dependent kinase 4, and its homologous deletion is common in human breast cancer. p16INK4 gene transfer has been reported to be efficacious in inducing growth inhibition of various human tumors such as brain, lung, prostate, and esophageal cancers. However, the efficiency of the p16INK4 gene with regard to growth inhibition of human breast cancer has not been studied extensively. To examine its tumor-suppressive function and its potential in breast cancer gene therapy, the wild-type p16INK4 gene was expressed in an adenovirus-mediated gene delivery system and introduced into breast cancer cell lines that do not express p16INK4 protein. Expression of the introduced p16INK4 blocked tumor cell entry into the S phase of the cell cycle, induced tumor cell apoptosis, and inhibited tumor cell proliferation both in vitro and in vivo. These results strongly suggest that p16INK4 is a tumor suppressor gene and suggest that it has potential utility in breast cancer gene therapy.

Combinational immunotherapy for established tumors with engineered tumor vaccines and adenovirus-mediated gene transfer.

There are currently extensive studies relating to cancer vaccines using tumor cells engineered to express immunogenes and cancer gene therapy using adenovirus (AdV)-mediated gene transfer. In this study, a mouse tumor cell line, VKCK, was cotransfected with genes coding for tumor necrosis factor-alpha (TNF-alpha) and costimulatory B7-1 molecule to enhance immunogenicity. The transfectant cell line VKCK-TNF-alpha/B7-1 showed reduced tumorigenicity and tumor regression. Its inoculation further induced protective immunity; both CD4+ and CD8+ T cells were involved in the induction phase, whereas only CD8+ T cells mediated the effector phase. Susceptible mice bearing VKCK tumors developed a T helper type 2-dominant response, whereas resistant mice with VKCK-TNF-alpha/B7-1 tumor regression developed a T helper type 1-dominant response to VKCK, indicating that the tumor regression was related to a shift in the cytokine profile of the host from type 2 to type 1. Vaccination of VKCK-TNF-alpha/B7-1 cells inhibited tumor formation derived from a single dose of 3 x 10(6) VKCK cells and eradicated 3-day tumors but not 10-day tumors. AdV-mediated TNF-alpha gene transfer by intratumoral injection of AdV-TNF-alpha significantly inhibited tumor growth but failed to eradicate any well-established tumors. However, combinational immunotherapy with vaccination of VKCK-TNF-alpha/B7-1 cells and AdV-mediated TNF-alpha gene transfer not only significantly inhibited tumor growth but also eradicated 10-day VKCK tumors in three of eight mice. Therefore, the present study may be useful not only in understanding the mechanisms responsible for an efficient antitumoral immunity, but also in establishing a more effective immunotherapeutic approach for cancer patients.

Regression of engineered tumor cells secreting cytokines is related to a shift in host cytokine profile from type 2 to type 1.

The precise role of the endogenous immune response in modulating cancer development remains unclear. In this study, three mouse tumor cell lines were used to elucidate the immune mechanisms for tumor regression versus tumor growth. These cell lines were (1) the poorly immunogenic VKCK cell line and (2) its two derived cell lines VKCK/RM4-tumor necrosis factor-alpha (TNF-alpha) and VKCK/RM4-interferon-gamma (IFN-gamma) engineered to secrete TNF-alpha and IFN-gamma, respectively. Our data showed that VKCK tumors grew aggressively in syngeneic BALB/c mice, and vaccination of irradiated VKCK cells failed to protect the mice from a subsequent challenge with the same tumor. In contrast, engineered VKCK tumor cells lost their tumorigenicity, and vaccination of engineered VKCK cells induced a protective immunity against VKCK cells that was mediated with VKCK-specific CD8+T cells. Susceptible mice developed a Th2-dominant response, whereas resistant mice developed a Th1-dominant response to VKCK. The T cell proliferative response and cytolytic activity against VKCK developed in both resistant and susceptible mice, but in the susceptible mice, these responses were much weaker compared with those in the resistant mice. Our results indicate that regression of tumor cells engineered to secrete cytokines TNF-alpha and IFN-gamma is related to a shift from a host type 2 to a type 1 cytokine profile. Our results further suggest that the failure of unmodified VKCK to generate efficacious T cells is not due to an inability to recognize tumor antigens but, rather, to the nature and magnitude of the antitumor immune response that develops. A better understanding of the mechanisms by which tumor cells modulate the host immune system may result in newer approaches for manipulating host-tumor interactions that favor the development of a protective antitumor immune response.

Recurrent pregnancy loss associated with endometrial hyperechoic areas (endometrial calcifications): a case report and review of the literature.

Endometrial calcifications occur sporadically and are associated with infertility. Previous uterine trauma during instrumentation and/or uterine infection are likely involved in their pathogenesis. The association between endometrial calcifications and recurrent pregnancy loss has been very infrequently reported. A 28-year-old woman with a history of two consecutive first trimester pregnancy losses presented with ultrasonographic hyperechoic endometrial areas associated with histologic endometrial calcification foci. A third pregnancy conceived before starting micronized oral progesterone supplementation also spontaneously aborted at eight weeks. During the fourth pregnancy, progesterone supplementation was taken for the initial 12 weeks. The endometrial lesions were no longer detectable and the pregnancy progressed to term without complications. Endometrial calcifications, related to intrauterine bone tissue, have been previously treated with curettage or with endoscopic surgery, and to the best of our knowledge, have not been reported to disappear spontaneously. In this case, regression of the endometrial calcifications and a favorable pregnancy outcome occurred in concert with oral micronized progesterone supplementation. A combination of transvaginal ultrasonography and endometrial biopsy appears to be an effective method for diagnosing and monitoring of this rare condition.

The spectrum of neuroendocrine differentiation among gastrointestinal carcinoids: importance of histologic grading, MIB-1, p53, and bcl-2 immunoreactivity.

CONTEXT: The advent of panneuroendocrine markers has helped to better depict the heterogeneity of gastrointestinal carcinoids. Consequently, it has been proposed that these tumors constitute a histologic spectrum that includes well-, moderately, and poorly differentiated carcinoids. However, the reproducibility of this grading system and its prognostic importance have sometimes been called into question. OBJECTIVE: To investigate the potential utility of cell proliferation and oncoprotein markers in augmenting the histologic classification. DESIGN AND SETTING: Retrospective study; tertiary care teaching hospital. METHODS: Fifty-eight patients with 41 well-differentiated, 12 moderately differentiated and 5 poorly differentiated carcinoids from various topographic sites of the gastrointestinal tract were selected and immunostained for panneuroendocrine markers, MIB-1, p53, and bcl-2. MAIN OUTCOME MEASURES: Degree of association between histologic grading, MIB-1, p53, and bcl-2 immunoreactivity and carcinoid metastatic behavior. RESULTS: The group comprised 30 males and 28 females whose mean age was 52.7 years (range, 14-81). Mean follow-up time was 85.8 months. All 58 patients tested positive for chromogranin A and/or synaptophysin. The group was divided into nonmetastatic (42/58, or 72.4%) and metastatic (16/58, or 27.6%) cases. Histologic grading tended to be associated with metastatic spread, but this occurrence of metastases did not attain statistical significance (P =.08). Positivity for MIB-1 (P =.004) and p53 (P =.04) was significantly associated with metastatic behavior, whereas bcl-2 was not (P = 0. 63). CONCLUSIONS: Although an organoid pattern and neuroendocrine immunophenotype help to define the spectrum of gastrotestinal carcinoids, this study suggests that the histologic grading of these tumors has some limitations with respect to predicting metastatic behavior. However, MIB-1 and p53 can compliment histologic grading as prognostic indicators in this regard while bcl-2 appears to be less useful.

Adenoviral transfer of xenogeneic MHC class I gene results in loss of tumorigenicity and inhibition of tumor growth.

The immune system confers protection against a variety of pathogens and contributes to the destruction of neoplastic cells. Foreign major histocompatibility complex (MHC) protein serves as a potent stimulus to the immune system. In this report, a mouse H-2Kb gene was introduced into two poorly immunogenic tumor cell lines, a mouse colonic carcinoma cell line, MCA-26 (H-2Kd), and a rat mammalian carcinoma cell line, LN-4, in an effort to stimulate tumor rejection. Our results showed that the expression of xenogeneic MHC class I antigen completely abolished the LN-4 tumorigenicity in rats, whereas the expression of allogeneic MHC class I antigen only partially reduced the MCA-26 tumorigenicity in mice. Rats with tumor regression of LN-4/H-2Kb developed a T helper type 1-dominant response, whereas rats with LN-4 tumor growth developed a T helper type 2-dominant response. The immunized rats that experienced LN-4/H-2Kb tumor regression further developed protective immunity against a subsequent challenge of LN-4 cells. This protective immunity was mediated by the LN-4 tumor-specific cellular immune response against both the transduced and the parental LN-4 cells. Recombinant adenoviral vectors are highly efficient at in vitro and in vivo gene delivery. The LN4 cells transfected with the recombinant adenovirus AdV-H-2Kb in vitro expressed the cell surface H-2Kb molecule by fluorescence-activated cell sorter analysis. Adenovirus-mediated H-2Kb gene transfer in vivo can further significantly inhibit pre-established LN-4 tumors. Those rats with complete tumor regression further developed protective immunity against the subsequent challenge of a parental LN-4 tumor. Therefore, our study indicates that the adenovirus-mediated transfer of xenogeneic MHC class I gene may be an effective alternative to the current protocol of cancer gene therapy in which the allogeneic MHC class I gene is used.

N-myristoyltransferase.

Myristoylation refers to the co-translational addition of a myristoyl group to an amino-terminal glycine residue of a protein by an ubiquitously distributed enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97). This review describes the basic enzymology, molecular cloning and regulation of NMT activity in various pathophysiological processes such as colon cancer and diabetes.

Enzyme-linked immunosorbent assay-based selection and optimization of elution buffer for TAG72-affinity chromatography.

An enzyme-linked immunosorbent assay (ELISA)-elution assay was developed to screen a large variety of elution buffers for selection of a suitable one for purification of the fusion protein FV/TNF-alpha by affinity chromatography. Various commonly used buffer systems utilizing widely differing conditions such as extreme pH, denaturants, chaotropic ions and polarity reducing reagents were investigated. Ammonia solution (1 M, pH 11.5) proved to exert the most suitable influence on dissociation of the FV/TNF-alpha/TAG72 complex while having a minimal protein denaturing effect on FV/TNF-alpha. The total yield of purified FV/TNF-alpha using the TAG72-affinity column with this elution system was 300-fold higher than that using the common elution buffer, 0.1 M glycine, 0.5 M NaCl, pH 2.7. Our study indicates that the ELISA-elution assay will be most useful in the selection of suitable elution buffers for affinity chromatography.

The impact of endoscopic technology on gastrointestinal pathology.

Since its introduction in the 1950s, fiberoptic endoscopy has dramatically altered the scope and practice of gastrointestinal (GI) pathology. Whereas examination by rigid instruments was generally restricted to the proximal digestive foregut and distal 25 cm of the large bowel, fiberoptic endoscopy extended these limits considerably, which resulted in a greater volume of biopsies submitted to the pathology laboratory. Furthermore, this technique is associated with a lesser degree of patient discomfort and a lower risk of complications compared to rigid or semiflexible endoscopy. In established endoscopy units, flexible endoscopy is performed increasingly with the videoscope rather than the fiberscope. With the added advantage of direct visualization, flexible endoscopy has eclipsed barium radiology as the premier investigative modality for GI diseases. Although upper GI endoscopy and colonoscopy account for the majority of biopsy material, there are other flexible endoscopic techniques, including endoscopic retrograde cholangiopancreatography and enterostomy. Flexible endoscopy has not only impacted the diagnosis of important disease entities (eg, reflux esophagitis, H. pylori gastritis, celiac disease and GI polyps and neoplasia), but it has also become a key component of surveillance protocols for dysplasia in Barrett's esophagus and idiopathic inflammatory bowel disease. Predicting major trends that may emerge from (GI flexible endoscopy in the future is somewhat difficult, but promising new avenues of investigation include increased use of endoluminal ultrasound and trans-bowel fine needle aspiration. Biopsy material will be submitted with more frequency for genetic molecular studies such as tumor development and progression and identification of infections agents; the priorities for handling biopsy material may have to be re-examined. Gastrointestinal (GI) biopsies constitute a substantial proportion of the surgical pathology load in most tertiary care medical centers. Based on topographic site of origin, the GI tract is the single largest component of the biopsy service in this institution. This relates in part to the high frequency of patients' complaints referable to the digestive tract and is also a result of the advances in GI endoscopy that have led to more widespread application of this technique. To gain a better appreciation of the impact of the changes in endoscopic techniques on gastrointestinal pathology, it is pertinent to examine the historical perspective from which the technology arose.

A case of peripartum eosinophilic myocarditis.

A 19-year-old postpartum patient with a previous history of asthma and eosinophilic myocarditis is described. Eosinophilic myocarditis is thought to be caused by exacerbation of the idiopathic hypereosinophilic syndrome by pregnancy. The diagnosis was made by a right ventricular endomyocardial biopsy, which showed an eosinophilic infiltrate with a few scattered foci of myonecrosis, but no fibrosis, vasculitis or granulomas. The patient's myocardial function continued to decline over a two-year follow-up period, despite normal levels of eosinophils. She developed echocardiographic evidence of diastolic and systolic dysfunction.

Asplenia as a cause of sudden unexpected death in childhood.

Sudden unexpected death in childhood is rare. The commonest causes of such deaths are a result of fulminating infections of the respiratory or nervous systems. Other causes include unsuspected congenital abnormalities of the heart, acute metabolic disorders, and rarities such as internal hemorrhages and pulmonary thrombosis. Recognition of children with congenital asplenia who are otherwise normal but have an increased susceptibility to overwhelming sepsis is extremely difficult. We reviewed 1763 autopsy files from our institution over 5 years (1990-1995), of which 293 were classified as pediatric cases. The vast majority of the cases were stillbirths and deaths within the first year of life as a result of complex congenital anomalies. Four cases of asplenia were identified in our entire series, 3 of which were of the congenital syndromal variety and 1 of which was a case of isolated sporadic congenital asplenia. All 4 cases of asplenia were analyzed in detail with respect to autopsy findings and cause of death. Severe complex cardiac malformations were present in the congenital syndromal asplenia patients; these other malformations contributed significantly to their death. In this report, we discuss in detail the autopsy findings in a previously healthy 4-year-old girl who presented with a brief 8-hour history of being unwell and died within 4 hours of admission into the hospital. She had sporadic, isolated congenital asplenia complicated by high-grade type 6B pneumococcemia and acute bilateral adrenal hemorrhage (Waterhouse-Friderichsen syndrome). Previously healthy children who clinically deteriorate very rapidly should have a blood smear done as part of their clinical workup. The detection of Howell-Jolly bodies on a peripheral blood smear can be an indicator of asplenia, and this diagnosis can be confirmed by medical imaging of the abdomen. Such steps may aid in the aggressive management of isolated congenital asplenia and thereby avert untimely death.

Adenovirus-mediated TNF-alpha gene transfer induces significant tumor regression in mice.

Recombinant adenovirus vectors are highly efficient at in vitro and in vivo gene delivery. The in vitro infection of a mouse colon adenocarcinoma cell line MCA-26 with the adenovirus AdV-LacZ can reach a maximal 75% of infectivity at an MOI of 1000. Intratumoral injection of AdV-LacZ (2X10(9) pfu) resulted in substantial gene transfer in nearly 70% of MCA-26 tumors. After the in vitro infection of AdV-TNF-alpha, infected MCA-26 cells showed significant secretion of TNF-alpha (45 ng/ml/10(6) cells) in tissue culture. The secretion peaks at day 2 and is diminished at day 4 following the viral infection. Infected MCA-26 tumor cells secreting TNF-alpha significantly reduced their tumorigenicity in syngeneic BALB/c mice. In mice bearing small tumors, intratumoral injection of 2X10(9) pfu of AdV-TNF-alpha virus with a repeated booster treatment resulted in complete regression of three tumors and significant diminution of the other two with a mean tumor-weight of 0.16 g; this is in contrast to 0.85 and 1.62 g for tumors injected with the control AdV-pLpA and PBS respectively (p < 0.01). Mice with complete tumor regression further developed protective immunity against the second challenge of MCA-26 inoculation. In mice bearing large tumors, this treatment also caused significant inhibition of tumor growth with a mean tumor weight of 0.65 g vis-a-vis 3.05 g for tumors injected with the control AdV-pLpA. On the contrary, in mice bearing large tumors, the treatment of tumors with pCI-TNF-alpha delivered by the gene gun did not induce significant tumor inhibition. These results indicate that the adenoviral delivery of TNF-alpha gene is more efficient than the particle-mediated gene gun device, and that adenovirus-mediated cytokine gene therapy may be a useful approach in the clinical management of human solid tumors.

Cytotoxic CD4+ T cells associated with the expression of major histocompatibility complex class II antigen of mouse myeloma cells secreting interferon-gamma are cytolytic in vitro and tumoricidal in vivo.

Vaccination of mouse myeloma V(K)C(K)-gamma (I) cells secreting interferon (IFN)-gamma and expressing enhanced major histocompatibility complex (MHC) class I antigen (Ag) resulted in protective immunity that was mainly mediated by CD8+ T cells. V(K)C(K)-gamma (I/II) cells expressing both enhanced MHC class I and class II Ags were isolated from V(K)C(K)-gamma (I) cells. These V(K)C(K)-gamma (I/II) cells were used to study the relationship between IFN-gamma secretion of tumor cells, its tumorigenicity, and its induced immunity, as well as to evaluate the cellular immunocomponents mediating this immunity. Our animal studies showed that IFN-gamma secretion by V(K)C(K)-gamma (I/II) cells curtailed its tumorigenicity in syngeneic BALB/c mice and further induced protective immunity against a subsequent graft of parental V(K)C(K) tumor. This immunity is mediated by both CD4+ and CD8+ T cells. The activation of CD4+ T cells is associated with enhanced expression of MHC class II Ag of V(K)C(K)-gamma (I/II) cells. These CD4+ T cells are tumor specific and cytolytic in an MHC-restricted fashion in vitro, and are tumoricidal in a T-cell adoptive transfer experiment in vivo. Our data thus demonstrate that vaccination of genetically modified tumor cells secreting IFN-gamma may provide beneficial antitumor effects by inducing both cytolytic CD4+ and CD8+ cytotoxic T lymphocytes, provided that these tumor cells express both enhanced MHC class I and class II Ags.

One hundred seventy-fold increase in excretion of an FV fragment-tumor necrosis factor alpha fusion protein (sFV/TNF-alpha) from Escherichia coli caused by the synergistic effects of glycine and triton X-100.

To target tumor necrosis factor alpha (TNF-alpha) to tumor cells, recombinant DNA techniques were used to construct and express the fused gene VKLVH-TNF-alpha, which encodes the secreted form of single-chain fusion protein sFV/TNF-alpha in Escherichia coli. sFV/TNF-alpha was secreted into the culture medium and purified by affinity chromatography. The production of the fusion protein in the culture medium under the optimal conditions of 30 degrees C and 37 micromol of isopropyl-beta-D-thiogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than that under the standard conditions of 37 degrees C and 1 mmol of IPTG per liter. Fusion protein excretion into culture medium with 2% glycine, 1% Triton X-100, or both of these two chemicals was either 14-, 38-, or 170-fold higher, respectively than that without the two chemicals. The final yield of sFV/TNF-alpha was estimated to be 50 mg/liter. The loss of integrity of the cellular membrane may be a potential mechanism for enhancement of fusion protein production and excretion by treatment with glycine and Triton X-100. This study thus provides a practical, large-scale method for more efficient production of the heterologous fusion protein sFV/TNF-alpha in E. coli by using glycine and Triton X-100.