Evidence of immunological activity in heterotropic autotransplanted splenic tissue in DBA/2 mice

After total splenectomy, a portion of the spleen was autotransplanted into subcutaneous ectopic sites of DBA/2 mice. These implanted fragments regenerated into splenic tissue that are microscopically indistinguishable from the structure of the original spleen. These implants are also immunologically functional as demonstrated by their ability to produce antibodies to sheep red blood cells (SRBC) and SRBC inhibition of splenic (implant) cell migration.     

Cells involved in cell-mediated and transplantation immunity in the rabbit. IV. The organ localization of the cells capable of migrating in vitro. Inhibition of migration by immunizing antigen

Rabbits were immunized with ovalbumin emulsified in complete Freund's adjuvant subcutaneously or with ovalbumin (OA) in saline intravenously. They were skin tested at intervals of time in order to determine the optimal sensitization time for the induction of the delayed skin reaction. The rabbits were also sacrificed and cell suspensions were prepared from the following organs: spleen, thymus, bone marrow, lymph nodes (popliteal), appendix, sacculus rotundus, and Peyer's patches. Peritoneal exudate cells were also obtained. These cell suspensions were tested for their ability to be inhibited in their migration in vitro by the specific sensitizing antigen. It was observed that the migration of all of the cell suspensions except for the bone marrow and peritoneal exudate cells could be inhibited by OA, but not by BGG, a non-cross-reacting antigen. Inhibition of migration was most marked at 3–4 weeks postsensitization and was negligible by 8–12 weeks, at a time when the delayed skin reaction was as extensive as in the early postsensitization period. Furthermore, the migration of cells of rabbits immunized with OA in saline intravenously was also markedly inhibited. It is concluded that, in the rabbit, different cell pathways are operative in the induction of the delayed skin reaction, on the one hand, and the facilitation of migration inhibition, on the other.     

Kinase-specific phosphorylation of the estrogen receptor changes receptor interactions with ligand, deoxyribonucleic acid, and coregulators associated with alterations in estrogen and tamoxifen activity

Posttranslational modifications of the estrogen receptor (ER) are emerging as important regulatory elements of cross talk between different signaling pathways. ER phosphorylation, in particular, has been implicated in the ligand-independent effects of ER and in tamoxifen resistance of breast tumors. In our studies, Western immunoblot analysis of endogenous ER in parental MCF-7 cells reveals specific, ligand-dependent phosphorylations at S118 and S167, with this ligand dependence being lost in tamoxifen-resistant, MCF-7 Her2/neu cells. Using highly purified components and sensitive fluorescence methods in an in vitro system, we show that phosphorylation by different kinases alters ER action through distinct mechanisms. Phosphorylation by Src and protein kinase A increases affinity for estradiol (E2), whereas ER phosphorylation by MAPK decreases trans-hydroxytamoxifen (TOT) binding. Affinity of ER for the consensus estrogen response element is also altered by phosphorylation in a ligand-specific manner, with decrease in affinity of MAPK- and Src-phosphorylated ER in the presence of TOT. ER phosphorylation by MAPK, AKT, or protein kinase A increases recruitment of steroid receptor coactivator 3 receptor interaction domain to the DNA-bound receptor in the presence of E2. Taken together, these results suggest that ER phosphorylation alters receptor functions (ligand, DNA, and coactivator binding), effecting changes that could lead to an increase in E2 agonism and a decrease in TOT antagonistic activity, reflecting changes encountered in tamoxifen resistance in endocrine therapy of breast cancer.     

Exploration of click reaction for the synthesis of modified nucleosides as chitin synthase inhibitors

Click reaction approach toward the synthesis of two sets of novel 1,2,3-triazolyl linked uridine derivatives 19a–19g and 21a–21g was achieved by Cu(I)-catalyzed 1,3-dipolar cycloaddition of 5′-azido-5′-deoxy-2′,3′-O-(1-methylethylidene)uridine (17) with propargylated ether of phenols 18a–18g and propargylated esters 20a–20g. Structure of one of the representative compound 19d was unambiguously confirmed by X-ray crystallography. Chitin synthase inhibition study of all these compounds 19a–19g and 21a–21g was carried out to develop antifungal strategy. Compounds 19d, 19e, 19f, and 21f were identified as potent chitin synthase inhibitors by comparing with nikkomycin. Compounds 19a, 19b, 19c, 19d, 21a, and 21b showed good antifungal activity against human and plant pathogens. Compounds 19a, 19b, 19f, 21c, 21f, and 21g were identified as lead chitin synthase inhibitors for further modifications by comparing results of inhibition of growth, % germ tube formation and chitin synthase activity.     

Astrocytes from familial and sporadic ALS patients are toxic to motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.     

Rejection of tumor metastases in Fischer 344 rats following the administration of killed Corynebacterium parvum

Summary Fischer 344 rats received subcutaneous grafts of syngeneic 13762 mammary adenocarcinoma cells. Thereafter, at 20 days, each animal of a group received either daily (×2) permeating intratumor injections of killed Corynebacterium parvum (1.0 mg) saline, and weekly (×4) intravenous (i.v.) or intraperitoneal (i.p.) C. parvum or saline. In addition, a group each (4 groups) of rats were treated with surgical extirpation of the growing tumor nodule and i.v. and i.p. C. parvum or saline administered at weekly (×4) intervals. The results revealed that following intratumor injection of C. parvum there was rejection of tumor by all animals which exhibited long-term survival for 29 months (intratumor saline: 0% survival). The groups of rats treated with surgical extirpation of the tumor and parenteral administration of C. parvum exhibited 70–75% survival for 20 months (saline treated: 20–35% survival). The surviving animals exhibited tumor-specific protection to subsequent tumor cell challenges. Macrophages separated from the lung, peritoneum, and spleen of rats from the intratumor C. parvum group exhibited respectively, 46.2%, 62.9%, and 29.4% cytotoxicity towards target 13762 tumor cells as measured by ⁵¹ Cr release. Similar studies using macrophages from the group treated with surgery and parenteral C. parvum revealed similar tumor cytotoxicity (pulmonary: 49.2%; peritoneal: 65.7%; spleen: 34.4%). The splenic lymphocytes from the intratumor and parenteral C. parvum groups exhibited, respectively, 16.9% and 14.7% ⁵¹ Cr release following incubation with target tumor cells.This study was supported in part by Grant No. CA18582-01 from the National Cancer Institute