[Pancreaticoduodenectomy combined with longitudinal pancreaticojejunostomy in the treatment of chronic pancreatitis: initial experience in 11 cases].

Objective: To investigate the surgical strategy for chronic pancreatitis complicated with suspected malignant lesions in the pancreatic head and pancreatolithiasis in the distal pancreas. Methods: This is a retrospective cohort study. Clinical data from 11 patients with chronic pancreatitis who underwent pancreaticoduodenectomy combined with longitudinal pancreaticojejunostomy(PD-L) were retrospectively collected(PD-L group) from the Department of Hepatobiliary Surgery of the First Affiliated Hospital of Xi'an Jiaotong University between December 2021 and September 2023. All patients were male with an age of (49.0±11.2) years(range:32 to 70 years). Their primary preoperative diagnoses included pancreatic lesions, chronic pancreatitis, pancreatolithiasis, and dilatation of the pancreatic duct. Data from 248 patients who underwent pancreaticoduodenectomy(PD) during the same period were retrospectively collected(PD group). There were 157 males and 91 females in the PD group, with an age of (61.5±10.8) years(range:27 to 82 years). Among them, 87 cases were diagnosed as pancreatic cancer or chronic pancreatitis. The propensity score matching method was used to reduce confounding bias between the two groups. The caliper value of 0.1 was used and the 1∶4 nearest neighbor matching method was used for the matching. Comparisons between the two groups were made using the independent sample t test, Mann-Whitney U test or χ2 test,respectively. Results: After complete excision of the specimen during pancreaticoduodenectomy, the key surgical step of PD-L was longitudinal pancreaticojejunostomy in the remaining pancreas. Intraoperative blood loss in the PD-L group was lower than that in the PD group [M(IQR)](300(200)ml vs. 500(500)ml, respectively; P<0.05). Similarly, hospitalization days(21.0(7.0)days vs. 25.0(8.5)days) and postoperative hospitalization days(13.0(8.0)days vs. 17.0(5.0) days) were also lower in the PD-L group compared to the PD group (P<0.05). There were no significant differences in the operation time and postoperative complication rate between the two groups(P>0.05). In the PD-L group, the postoperative follow-up time was 5(5)months(range: 3 to 21 months). One case was lost for follow-up. Abdominal pain was relieved in 10 patients. Additionally, abdominal distension and steatosis were alleviated in 8 cases. Furthermore, 5 cases of diabetes mellitus showed improved control of HbA1c and fasting blood glucose levels after surgery. Conclusions: PD-L treatment can be used to treat chronic pancreatitis complicated by suspected malignant lesions in the pancreatic head and pancreatolithiasis in the distal pancreas. PD-L also has advantages in removing stones from the pancreatic duct and evaporation of pancreatic fluid. However, due to the single-center design and the small sample size of this study, further practice and long-term follow-up are still necessary.

[Effect of pancreatic extracorporeal shock wave lithotripsy on chronic pancreatitis stones].

Objective: To analyze the therapeutic effect and safety of pancreatic extracorporeal shock wave lithotripsy(P-ESWL) for patients with chronic pancreatitis complicated by stones of the pancreatic duct and to investigate the influencing factors. Methods: A retrospective analysis was performed on clinical data from 81 patients with chronic pancreatitis complicated by pancreatic duct calculus treated with P-ESWL in the Department of Hepatobiliary Surgery, the First Affiliated Hospital of Xi 'an Jiaotong University from July 2019 to May 2022. There were 55 males(67.9%) and 26 females(32.1%). The age was (47±15)years (range: 17 to 77 years). The maximum diameter(M(IQR)) of the stone was 11.64(7.60) mm, and the CT value of the stone was 869 (571) HU. There were 32 patients (39.5%) with a single pancreatic duct stone and 49 patients(60.5%) with multiple pancreatic duct stones. The effectiveness, remission rate of abdominal pain, and complications of P-ESWL were evaluated. Student's t test, Mann Whitney U test, χ2 test, or Fisher's exact test was used to compare the characteristics between the effective and ineffective groups of lithotripsy. The factors influencing the effect of lithotripsy were analyzed by univariate and multivariate logistic regression analysis. Results: Eighty-one patients with chronic pancreatitis were treated with P-ESWL 144 times, with an average of 1.78 (95%CI:1.60 to 1.96) times per person. Among them, 38 patients(46.9%) were treated with endoscopy. There were 64 cases(79.0%) with effective removal of pancreatic duct calculi and 17 cases(21.0%) with ineffective removal. Of the 61 patients with chronic pancreatitis accompanied by abdominal pain, 52 cases(85.2%) had pain relief after lithotripsy. After lithotripsy treatment, 45 patients(55.6%) developed skin ecchymosis, 23 patients(28.4%) had sinus bradycardia, 3 patients(3.7%) had acute pancreatitis, 1 patient(1.2%) had a stone lesion, and 1 patient(1.2%) had a hepatic hematoma. Univariate and multivariate logistic regression analysis showed that the factors affecting the efficacy of lithotripsy included the age of patient(OR=0.92, 95%CI: 0.86 to 0.97), the maximum diameter of the stone(OR=1.12,95%CI:1.02 to 1.24) and the CT value of the stone(OR=1.44, 95%CI: 1.17 to 1.86). Conclusions: P-ESWL is effective in the treatment of patients with chronic pancreatitis complicated by calculi of the main pancreatic duct.Factors affecting the efficacy of lithotripsy include patient's age, maximum stone diameter, and CT value of calculi.

Distinct Kondo Screening Behaviors in Heavy Fermion Filled Skutterudites with 4f

CeOs_{4}Sb_{12} (COS) and PrOs_{4}Sb_{12} (POS) are two representative compounds that provide the ideal vantage point to systematically study the physics of multi-f-electron systems. COS with Ce 4f^{1}, and POS with Pr 4f^{2} configurations show distinct properties of Kondo insulating and heavy fermion superconductivity, respectively. We unveiled the underlying microscopic origin by angle-resolved photoemission spectroscopy studies. Their eV-scale band structure matches well, representing the common characters of conduction electrons in ROs_{4}Sb_{12} systems (R=rare earth). However, f electrons interact differently with conduction electrons in COS and POS. Strong hybridization between conduction electrons and f electrons is observed in COS with band dependent hybridization gaps, and the development of a Kondo insulating state is directly revealed. Although the ground state of POS is a singlet, finite but incoherent hybridization exists, which can be explained by the Kondo scattering with the thermally excited triplet crystalline electric field state. Our results help us to understand the intriguing properties in COS and POS, and provide a clean demonstration of the microscopic differences in heavy fermion systems with 4f^{1} and 4f^{2} configurations.

Analysis of long non-coding RNA expression profiles in disuse osteoporosis using microarray and bioinformatics.

Disuse osteoporosis (DOP) is one of the major consequences of long space flights. DOP also occurs in patients with spinal cord injuries and prolonged bedridden states that can have a severe impact on human health. Bone marrow mesenchymal stem cells (BMSCs) are multipotent stromal cells that play an important role in bone homeostasis. Long non-coding RNAs (lncRNAs) are involved in regulating osteogenic differentiation of BMSCs, and their abnormal expression might lead to the formation of orthopedic diseases. However, the specific mechanism of DOP has not yet been elucidated. All sequencing data were obtained from Gene Expression Omnibus (GEO) datasets. The limma package of R was applied to identify DEmRNAs and DElncRNAs. Pearson correlation coefficients (PCC) between DElncRNADEmRNA expression levels were calculated. Functional annotation was performed for DEmRNAs coexpressed with DElncRNAs. In addition, the Cytohubba plug-in in Cytoscape was applied to determine the top 10 hub genes. Finally, connectivity map (CMap) analysis was used to identify potential therapeutic drugs for DOP. The gene expression data, GSE100930 and GSE17696, were retrieved from the GEO database. A total of 2,212 differentially expressed mRNAs (DEmRNAs) and 22 differentially expressed lncRNAs (DElncRNAs) were obtained. Gene ontology (GO) functional terms, Kyoto Encyclopedia of Genes, and Genomes (KEGG) pathway enrichment analysis reveal 30 significant GO terms and 13 significant pathways. A coding-non-coding gene co-expression (CNC) network was constructed to study the potential role of hub-DElncRNAs and their co-expressed DEmRNAs in DOP. The lncRNAs, GSNAS1, SNHG12, and EPB41LA4A-AS1, were significant in the CNC network and potential regulators of DOP development. Three bioactive compounds (scoulerine, kinetin riboside, dexanabinol) with potential therapeutic significance for DOP were obtained through the Connectivity Map (CMAP) analysis. Our study revealed a new mechanism for a lineage shift of bone marrow mesenchymal stem cells under microgravity, and linked the function of protein-coding mRNAs with ncRNAs, which may contribute to the development of new therapies for DOP.

In Situ Measurement of Vacuum Window Birefringence using 25Mg+ Fluorescence.

Accurate control of the polarization states of laser light is important in precision measurement experiments. In experiments involving the use of a vacuum environment, the stress-induced birefringence effect of the vacuum windows will affect the polarization states of laser light inside the vacuum system, and it is very difficult to measure and optimize the polarization states of the laser light in situ. The purpose of this protocol is to demonstrate how to optimize the polarization states of the laser light based on the fluorescence of ions in the vacuum system, and how to calculate the birefringence of vacuum windows based on azimuthal angles of external wave plates with Mueller matrix. The fluorescence of 25Mg+ ions induced by laser light that is resonant with the transition of |32P3/2,F = 4, mF = 4 â†’ |32S1/2,F = 3, mF = 3 is sensitive to the polarization state of the laser light, and maximum fluorescence will be observed with pure circularly polarized light. A combination of half-wave plate (HWP) and quarter-wave plate (QWP) can achieve arbitrary phase retardation and is used for compensating the birefringence of the vacuum window. In this experiment, the polarization state of the laser light is optimized based on the fluorescence of 25Mg+ ion with a pair of HWP and QWP outside the vacuum chamber. By adjusting the azimuthal angles of the HWP and QWP to obtain maximum ion fluorescence, one can obtain a pure circularly polarized light inside the vacuum chamber. With the information on the azimuthal angles of the external HWP and QWP, the birefringence of the vacuum window can be determined.

A simple method for in situ measurement of vacuum window birefringence.

We present a simple method to measure the degrees of circular polarization (DoCP) of laser light inside a vacuum chamber and the birefringence of a vacuum window by detecting the fluorescence emitted by Doppler cooled ions in an ion trap. Imperfect laser polarization will cause ions to be pumped to the dark state which will decrease the fluorescence rates of the ions. With a simulation based on the rate equations of the relevant energy levels of 25Mg+ ions, we find that the fluorescence rate is sensitive to the DoCP of the laser. Based on the simulation result, we present a new method to optimize the DoCP of the laser inside the vacuum chamber by adjusting fast axis azimuthal angles of a half-wave plate and a quarter-wave plate outside the vacuum chamber. The laser light is optimized to be circularly polarized with an uncertainty of the DoCP of 7.8 × 10-5. With the obtained polarization information on both sides of the vacuum window and treating the vacuum window as an unknown wave plate, the phase delay and the fast axis azimuthal angle of the vacuum window can be determined in the form of Mueller matrix. The phase delay is determined to be 197.60(39)°, and the fast axis azimuthal angle is determined to be 104.00(5)°.

Correlation of natural killer activity with tumorigenesis of a preneoplastic mouse mammary lesion.

Tissue infiltrating lymphocytes isolated from the preneoplastic mouse mammary hyperneoplastic alveolar nodule (HAN) tissue line C4 express high levels of natural killer (NK) activity, which gradually wanes as spontaneous tumors develop (W. Z. Wei and G. Heppner. Br. J. Cancer, 55: 589-594, 1987). Experiments were performed to test whether modulation of NK cell activity would be associated with altered progression of HAN to tumor. Administration of polyinosinic-polycytidylic acid, which activates NK activity but does not directly affect mammary epithelial cell growth, to HAN-bearing mice enhanced tumor progression, as measured by a decrease in the latency period and increase in the incidence of mammary adenocarcinomas developing in the HAN implants. Antiasialo GM1, which reduces NK activity, reduced tumor progression. The net effect of indomethacin, which may inhibit mammary epithelial cell growth but enhances NK cell function, was to prolong the latency period of tumor development. However, this effect was reversed by interleukin 2, which activates NK cells. These findings suggest that NK activity may provide positive signals for progression of preneoplastic mammary lesions to frank neoplasia.

Characterization of lymphocytic infiltrates in normal, preneoplastic, and neoplastic mouse mammary tissues.

Lymphocytic infiltrates were isolated from normal, preneoplastic, and neoplastic mouse mammary tissues. The surface markers on the infiltrating lymphocytes were characterized by immunofluorescent staining and flow cytometry. Preneoplastic and neoplastic tissues contained 10- to 20-fold more in situ lymphocytes than did the normal pregnant gland. Most of these lympocytes were T-cells. Relative to the T-cells in normal gland, the T-cells in C4 preneoplastic hyperplastic alveolar nodules and their spontaneous tumors have shifted in favor of the killer-suppressor subpopulation. This shift of T-cell subpopulations was a localized phenomenon and was not seen in the lymph nodes of hyperplastic alveolar nodules and tumor bearing mice. C4 lesion infiltrating cells also contained a subpopulation of lymphocytes that expressed 5- to 6-fold more LFA-1 antigen (lymphocyte function associated antigen-1) than did normal lymph node cells. The infiltrating lymphocytes of mammary tumors from cloned cell lines, on the contrary, had the same staining profile as did the lymphocytes from normal gland. Since most studies with human breast cancer infiltrates have demonstrated increased killer/suppressor T-cells and the presence of activated lymphocytes (J. Hurlimann and P. Saraga, Int. J. Cancer, 35: 753-762, 1985; H.L. Whitwell, H.P.A. Hughes, M. Moore, and A. Ahmed, Br. J. Cancer, 49: 161-172, 1984; and J.A. Ledbetter, R.V. Rouse, H. Spedding Micklem, and L. Herzenberg, J. Exp. Med., 152: 280-295, 1980) the C4 hyperplastic alveolar nodules and spontaneous tumor system may be a more relevant model for studying breast cancer infiltrates.

Adoptive transfer of V beta 2-deleting activity with host cells from mice implanted with C4 preneoplastic hyperplastic alveolar nodules.

Selective deletion of mature peripheral V beta 2+ T-cells was observed in BALB/c mice implanted with the syngeneic C4 preneoplastic hyperplastic alveolar nodule (HAN) but not in mice with sham surgery (W. Z. Wei, R. Ficsor-Jacobs, S. J. Tsai, and R. Pauley, Cancer Res., 51:3331-3333, 1991). We now report the participation of host cells in that process. Peripheral V beta 2+ T-cells were reduced by 50% or more 4 weeks after an i.v. injection of 10 x 10(6) spleen cells from C4 HAN-bearing mice. Both T- and B-cell-enriched splenocytes mediated V beta 2 deletion. Secondary adoptive transfer of splenocytes from the recipients of the primary adoptive transfer also resulted in V beta 2+ T-cell deletion. The splenocytes lose V beta 2-deleting activity after 500 rad irradiation. V beta 2 deletion induced by either C4 HAN or splenocytes was more profound in CD4+ than in CD8+ T-cells. Loss of V beta 2+CD4+ T-cells was observed 5 days after the adoptive transfer of splenocytes, whereas V beta 2+ CD8+ cells were not reduced until day 9 or later. The differential rate of V beta 2+ CD4+ and CD8+ T-cell reduction continued for at least 7 weeks after the adoptive transfer. The pattern of V beta 2 deletion and the sequence of T-cell loss is similar in the recipients of C4 HAN or of adoptively transferred splenocytes. Southern blot analysis demonstrates non-germ line Mtv or MMTV proviral DNA in C4 HAN. Splenocytes of C4 HAN-bearing mice express a higher level of 1.7-kilobase long terminal repeat transcript than normal BALB/c splenocytes, suggesting a role for a unique Mtv/MMTV provirus in V beta 2 deletion.

Elimination of V beta 2 bearing T-cells in BALB/c mice implanted with syngeneic preneoplastic and neoplastic mammary lesions.

The effect of growth of BALB/c C4 preneoplastic and neoplastic mammary lesions on the host T-cell repertoire was investigated. T-cells with specific V beta regions (V beta 2, -6, -8.1-2, -11, and -14) in the T-cell receptors were enumerated in C4 hyperplastic alveolar nodule (HAN)-infiltrating lymphocytes and lymph node cells from both C4 HAN and C4 tumor-bearing mice by flow cytometry with V beta-specific monoclonal antibodies. Growth of C4 HAN and tumor induced selective deletion of peripheral V beta 2+ T-cells. Elimination of V beta 2+ T-cells [7.2 +/- 0.8% (SD) of normal lymph node T-cells] by C4 HAN was biphasic and irreversible. Approximately one-half of the V beta 2+ T-cells were lost within the first month of C4 HAN implantation. Further reduction of V beta 2+ T-cells took place after another 3 months, at which time the level of V beta 2 was reduced to 1.2 +/- 0.1% of the total T-cell population. V beta 2 deletion occurred in BALB/c mice which had been implanted with C4 HAN at either 3 weeks or 2 months of age. Loss of V beta 2+ T-cells was not reversed by removal of C4 HAN. C4 tumor also induced V beta 2+ T-cell deletion. These results demonstrate a novel V beta 2 deleting activity expressed by C4 mammary lesions and suggest that during mouse mammary tumorigenesis, a unique "superantigen" is expressed which can cause profound systemic changes in the T-cell repertoire.

Activation of natural killer cells by mouse mammary tumor virus C4 in BALB/c and T-cell receptor V beta 2-transgenic mice.

Lymphocytic infiltrates of BALB/c C4 hyperplastic alveolar nodule (HAN) have elevated natural killer (NK) activity, which correlates positively with the progression of C4 HAN to tumor: C4 HAN produces an infectious mouse mammary tumor virus, MMTV(C4), which encodes a superantigen that activates and deletes T-cells with the V beta 2 segment in the T-cell receptor. In this report, NK activation by both MMTV(C4) and MMTV(C4) superantigen was tested. NK activity was measured in naive BALB/c mice, BALB/c mice depleted of V beta 2+ T-cell, or V beta 2-transgenic mice after they received injections of either purified MMTV(C4) or MMTV(C4)-infected splenocytes. Elevated NK activity was observed in BALB/c mice receiving MMTV(C4) or MMTV(C4)-infected splenocytes. Depletion of V beta 2+, but not V beta 8+, T-cells by specific anti-V beta hybridoma before injection of MMTV(C4)-infected cells reduced but did not eliminate NK activation. NK activation in V beta 2-transgenic mice occurred before massive CD4 T-cell deletion took place and was more pronounced than that in the nontransgenic littermates. These results indicate that MMTV activates NK cells through superantigen-dependent and -independent pathways and supports the role of MMTV(C4) in the augmented NK activity observed in C4 HAN infiltrates. The progression of C4 HAN to tumor represents a model system for the analysis of how tumorigenesis may be affected by lesion-associated viruses.

Expression of epithelial-like markers and class I major histocompatibility antigens by a murine carcinoma growing in the mammary gland and in metastases: orthotopic site effects.

A spontaneous murine mammary carcinoma, designated SP1, grew more aggressively in the mammary gland than in the subcutis exhibiting a 10-fold lower 50% lethal tumor dose and the ability to metastasize spontaneously from the orthotopic mammary gland site. The appearance of metastasis could be abrogated by resection of the primary tumor up to 21 days postinjection, arguing against the possibility that metastasis occurred due to trauma of the injection and/or healing processes. In addition, tumor cells recovered from lung metastases exhibited an increased ability to metastasize when reinjected into either the s.c. or mammary sites. Tumor cells from lung metastases showed low levels of Class I major histocompatibility (MHC) antigens, like the parental SP1 cells, but were found to express differentiation markers typical of normal basal and luminal mammary epithelium. SP1 tumors expressed increased Class I MHC antigens, as well as high levels of basal and luminal breast epithelial markers, within 7 days of implantation into the mammary gland. On the other hand, SP1 tumors growing in the subcutis never expressed increased Class I MHC levels and expressed the epithelial marker antigens at lower levels and not until at least 21 days of growth. Removal of host epithelium by cauterization of the mammary bud at 3 weeks had no effect on the increased growth, metastasis and acquired heterogeneity of MHC and epithelial associated antigens, suggesting that the mammary gland stroma was responsible for the observed phenomenon. These findings suggest that the mammary gland either selects distinct tumor subpopulations, or induces a phenotypic change leading to tumor progression and the generation of metastatic subpopulations.

Monoclonal antibodies directed against preneoplastic and neoplastic murine mammary lesions.

We have produced a panel of monoclonal antibodies directed against a dimethylbenzanthracene-induced murine mammary tumor. Five rat-mouse hybridomas produced antibodies that bound to some murine mammary tumors, but not to normal renal adherent cells, lymphocytes, 3T3 fibroblasts, red blood cells, or mammary gland. One of these antibodies, designated AMT8, was selected for further evaluation based on its relatively strong reactivity, as determined by immunofluorescence. Indirect immunofluorescent studies on frozen histological tissue sections and quantitative immunofluorescent binding studies on cultured normal and tumor cells revealed that AMT8 was bound to certain murine mammary tumors and their preneoplastic hyperplastic nodules, but not to normal murine organs including normal mammary glands. Two tumors and their hyperplastic alveolar nodule counterparts that contained the antigen recognized by AMT8 did not express functional estrogen and progesterone receptors, indicating that antigen expression was not dependent on functional receptors. The antigen recognized did not cap, was found to modulate slowly, and was reexpressed in the presence of excess AMT8. From these findings, we conclude that AMT8 may prove to be a valuable tool for the study of early mammary tumorigenesis.

Cellular immunity to the Her-2/neu protooncogene.

Her-2/neu (HER-2) is a 185-kDa receptor-like glycoprotein that is overexpressed by a variety of tumors such as breast, ovarian, gastric, and colorectal carcinomas. Overexpression of this oncogene is directly associated with malignant transformation of epithelial cells. The frequency of HER-2 overexpression varies among the different types of cancers, but universally represents a marker of poor prognosis. The critical role of HER-2 in epithelial oncogenesis as well as its selective overexpression on malignant tissues makes it an ideal target for immunotherapy. Antibodies and T cells reactive to HER-2 are known to naturally occur in patients with HER-2 positive tumors, confirming the immunogenicity of the molecule. Both antibodies as well as T cells reactive to HER-2 have been utilized for immunotherapy of HER-2 positive tumors. The "humanized" monoclonal antibody Herceptin has been tested in several clinical trials and found to be an effective adjuvant therapy for HER-2 positive breast and ovarian cancer patients. However, the frequency of patients responding to Herceptin is limited and a majority of patients initially responding to Herceptin develop resistance within a year of treatment. The use of vaccination strategies that generate T cell responses with or without accompanying antibody responses may serve to mitigate the problem. Various strategies for generating T cell-mediated responses against HER-2 are currently being examined in animal models or in clinical trials. The potential advantages of the various approaches to immunotherapy, their pitfalls, and the mechanisms by which HER-2 positive tumors can evade immune responses are discussed in this review.

Inflammatory infiltrates of experimental mammary cancers.

The purpose of this review was to summarize observations on the type and function of inflammatory infiltrates of mouse mammary tumors and to speculate on the underlying mechanisms and the significance of infiltrates to mammary tumor biology. Although the major conclusion is that much more work is needed, certain themes seem to be emerging. The number of infiltrating cells can be very high but is unrelated to biological behavior of the tumors. What seems to be important is the relative contributions of inflammatory cell subsets. In the case of T-cell subsets and NK cells, the infiltrates from tumors of long-term cell lines so far seem uninformative. The general characteristics are similar to those of infiltrates from rapidly proliferating, normal mammary tissues. These characteristics do not correlate with diverse biological behavior or malignant potential. A more informative model appears to be one in which the development of tumors from preneoplastic tissue can be observed. Here our attention is currently focused on NK cells. By contrast, the correlation between activated TAM and metastatic behavior suggests that our transplantable MMT lines may be biologically relevant in the study of infiltrating macrophages. We are especially interested in the role of TAM in the generation of tumor cell variability. Overall, our data indicate that the host infiltrate is another manifestation of both inter- and intra-tumor heterogeneity and, as such, is not simply a response to, but, rather, a part of the tumor ecosystem. Unraveling the cellular and molecular mechanisms that govern the inflammatory cell component of tumors should provide insight into the types of cellular interactions that result in tumor development and progression.

PEPMOTIF: a program for locating class I major histocompatibility complex restricted peptides in protein sequences.

PEPMOTIF is a computer program which analyzes protein sequences for the occurrence of peptides up to ten residues in length which contain motifs presented by particular class I major histocompatibility complexes. Any peptide motifs defined by the user can be identified in a protein sequence of interest. PEPMOTIF generates a listing of all motif-containing peptides found in the protein, and two modes of data output are provided: (1) direct printout, or (2) storage in a text file on disk.

A new assay to measure monoclonal antibody-dependent complement or macrophage-mediated tumor growth inhibition.

A new in vitro assay has been developed to measure the inhibition of tumor growth by antibody and complement or by antibody and macrophages. Tumor cells (1 X 10(5) cells) or a mixture of tumor cells (1.5 X 10(4) cells) and macrophages (1.5 X 10(5) cells) are immobilized in a 1 microliter agarose droplet. Antibody is added to the medium bathing the agarose droplet. Complement is also added to wells containing droplets with tumor cells alone. The area covered by tumor cell monolayer is measured non-destructively from day 0 to day 7 with a split image tracing device. Cell growth is expressed by the increase in the square root of the measured area. This assay does not require isotopes and can be used to test tumor cells freshly dissociated from solid tissues. It permits cell-cell interactions which may change the sensitivity of tumor cells to various treatments. The extended period of observation allows the testing of multiple treatments. Surviving cells can also be recovered for further study. This assay may be useful for testing the efficacy of monoclonal antibody treatment on solid tumors.

Inhibition of tumor growth by peptide specific cytotoxic T lymphocytes in a three-dimensional collagen matrix.

Tumor associated, MHC I restricted antigenic peptides have been identified in both human and mouse tumors. Cytotoxic T lymphocytes (CTL) which recognize these tumor associated antigenic peptides are potential anti-cancer effectors. The anti-tumor activity of CTL is usually measured in vitro by the 51Cr release assay and in mice by tumor growth inhibition which is the most direct assessment of anti-tumor effect. In clinical studies, an in vivo tumor growth inhibition assay is not an option and an in vitro assay which corroborates with in vivo tumor growth is needed to assess the long-term outcome of CTL activity. Here, a three-dimensional (3-D) collagen gel assay was developed to measure in vitro the inhibition of mouse mammary tumor growth by anti-tumor CTL. BALB/c mouse CTL were induced with peptide E474 SFAVATTAL which was expressed by mouse mammary tumor cells D2F2. To measure D2F2 tumor growth inhibition in vitro, a mixture of tumor cells and anti-E474 CTL in a 1 microl cell bolus was embedded in the collagen gel. Complete eradication of tumor growth was observed at E:T ratio of or greater than 1:1. rIL-2 supplementation was necessary to achieve long-term tumor growth inhibition. Even spontaneous D2 tumor explant could be grown in the collagen gel and addition of anti-E474 to this culture reduced tumor growth. This assay system provides a realistic and sensitive alternative to the in vivo tumor growth inhibition assay and allows easy adaptation to test additional therapeutic reagents.

Improved anti-tumor reactivity with monoclonal anti-idiotypic antibody conjugated to syngeneic mouse red blood cells.

An anti-idiotypic (anti-Id) antibody (Ab), by resembling an epitope on an antigenic molecule may serve as a surrogate antigen and induce specific immune reactivity to the natural antigen. Anti-Id Ab are often conjugated to a foreign protein, such as keyhole limpet hemocyanin (KLH) to elicit a stronger response. However, KLH, a potent antigen itself, may provoke unnecessary and undesirable immune reactivity. In this study, syngeneic mouse red blood cells (RBC) were tested as carriers for anti-Id immunization. In a preliminary experiment, BALB/c mice were immunized with normal rat immunoglobulin G (NRIg)-MRBC or NRIg-KLH. Anti-NRIg responses induced by 2-0.2% of NRIg-MRBC (4-0.4 micrograms of NRIg) superseded those induced by NRIg-KLH (50 micrograms of NRIg). Immunization with NRIg-MRBC did not result in autoreactivity against self antigens on autologous RBC. Monoclonal antibody 2F10, which resembles an epitope on mouse mammary tumor virus (MMTV) envelop protein Gp52, was conjugated to MRBC and used as a surrogate antigen for Gp52. Immune sera were tested in cytotoxicity assay against MMT 68H which expresses Gp52. Between 2-4 weeks after the immunization, specific cytotoxic antibodies were detected in mice immunized with 2.0%, 0.2% and 0.02% 2F10-MRBC, with 0.2% 2F10-MRBC being the most effective dose. 2F10 immune sera did not react with autologous RBC. Therefore, MRBC are at least as effective, and may be more effective than KLH as carriers for Ig immunization. Repeated treatment will be feasible since MRBC are not immunogenic.

Systematic identification of H-2 Kd binding peptides and induction of peptide specific CTL.

Most peptides with putative MHC I restricted sequence motifs do not bind to the corresponding MHC I nor induce cytolytic T cells. There exist additional constraints which limit peptide binding and immunogenicity. To identify immunogenic peptides in novel protein sequences, it will be necessary to first evaluate peptide binding to MHC I. In this study, a soluble single chain fusion protein SC-Kd was used to evaluate potential Kd binding peptides from the sequences of mouse mammary tumor virus gag and env proteins. A total of 27 peptides were identified which displayed the reported Kd restricted motif. Of the 27 peptides, six demonstrated strong to moderate binding to SC-Kd. The strongest binding peptides expressed tyrosine or phenylalanine at position 2 and leucine at the C-terminus. The capability of MMTV peptides to induce CTL corresponds to their SC-Kd binding activity. Of the six peptides that demonstrated moderate to strong binding, five induced CTL in BALB/c mice. These peptides induced CTL after 1-3 in vivo immunizations followed by 5 day in vitro stimulation. Furthermore, a single in vitro stimulation of naive lymphocytes with strong-binding G425 was sufficient to induce significant CTL activity. Weak or non-binding peptides did not induce CTL. Therefore, peptide binding to SC-Kd is a predictive indicator of CTL inducing activity.