In vivo resistance of secondary antitumor immune response to cyclophosphamide: effects on T cell subsets

Summary We have analyzed the effects of high doses of cyclophosphamide (Cy) on primary and secondary antitumor immune response against immunogenic (tum^–) variants of Lewis lung carcinoma (3LL) treated in vitro with UV light. Normal mice and mice previously immunized with tum^– clones were inoculated i.p. with Cy (200 mg/kg body weight) and 24 h later challenged intrafootpad with tum^– or parental 3LL cells. Cy treatment suppressed the primary immune response of normal animals and allowed the growth of tum^– cells. In contrast, Cy-treated immune mice rejected the tumor challenge. The in vivo treatment with Cy decreased the total number of lymphoid cells in the spleens, as well as the proportion of B lymphocytes; however, it increased the percentage of both Lyt2⁺ and L3T4⁺ lymphocytes. Thus, the immunosuppressive effects of Cy on the primary antitumor response could not be attributed to elimination of major T lymphocyte subpopulations. Although the treatment of immune mice with Cy did not significantly impair their antitumor resistance, nor the proportion of Lyt2⁺ and L3T4⁺ lymphocytes in their spleens, the in vitro generation of cytotoxic T lymphocytes (CTL) was markedly reduced.After Cy treatment, the proliferative ability of spleen cells in response to interleukin-2 (IL-2) was substantially impaired. Using monoclonal antibodies to the IL-2 receptor, we found that Cy-treated T lymphocytes failed to fully express the IL-2 receptor following in vitro stimulation with irradiated tumor cells. In line with these findings, the in vitro generation of CTL was not restored by addition of recombinant IL-2 to the cultures. In vivo experiments using purified functional subsets of immune T cells showed that Lyt1⁺, but not Lyt2⁺ lymphocytes were able to transfer antitumor immunity in normal irradiated recipients.Therefore, since Ly1⁺ T lymphocytes were responsible for the antitumor resistance in vivo, the Cy-induced impairment of CTL generation did not affect the ability of immune mice to reject a secondary tumor challenge.This project has been funded at least in part with Federal funds from the Department of Health and Human Services, under contract number NO1-CO-23910 with Resources, Inc. The contents of this publication do not necessarily reflect the view or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government     

Diversity-oriented synthesis of a cytisine-inspired pyridone library leading to the discovery of novel inhibitors of Bcl-2

Four enantiopure cytisine-inspired scaffolds can be accessed via a versatile pyrrolidine template derived from a stereocontrolled [3+2] azomethine ylide–alkene cycloaddition. Differential ester protection allows for the selective formation of either a bridged bicyclic or tricyclic scaffold via pyridone cyclization. Solid-phase diversification of the pyridone scaffolds yielded a diverse library of 15,000 compounds enabling the discovery of a novel class of Bcl-2 inhibitors.     

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death¹. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37°C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.Open in a separate windowClick here to view.^{(36M, flv)}     

Better assessment of physical function: item improvement is neglected but essential

Introduction  

Physical function is a key component of patient-reported outcome (PRO) assessment in rheumatology. Modern psychometric methods, such as Item Response Theory (IRT) and Computerized Adaptive Testing, can materially improve measurement precision at the item level. We present the qualitative and quantitative item-evaluation process for developing the Patient Reported Outcomes Measurement Information System (PROMIS) Physical Function item bank.     

Stems and Standards: Social Interaction in the Search for Blood Stem Cells

This essay examines the role of social interactions in the search for blood stem cells, in a recent episode of biomedical research. Linked to mid-20th century cell biology, genetics and radiation research, the search for blood stem cells coalesced in the 1960s and took a developmental turn in the late 1980s, with significant ramifications for immunology, stem cell and cancer biology. Like much contemporary biomedical research, this line of inquiry exhibits a complex social structure and includes several prominent scientific successes, recognized as such by participating researchers. I use personal interviews and the published record to trace the social interactions crucial for scientific success in this episode. All recognized successes in this episode have two aspects: improved models of blood cell development, and new interfaces with other lines of research. The narrative of the search for blood stem cells thus yields a robust account of scientific success in practice, which generalizes to other scientific episodes and lends itself to expansion to include wider social contexts.     

Dual Effects of Anandamide on NMDA Receptor-Mediated Responses and Neurotransmission

Abstract: Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as Δ⁹-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (ΔCa²⁺_NMDA) in rat brain slices. The presence of anandamide reduced ΔCa²⁺_NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting ΔCa²⁺_NMDA, they also revealed another, underlying mechanism by which anandamide influences ΔCa²⁺_NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated ΔCa²⁺_NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.     

Anti-oxidative and anti-inflammatory activities of two different species of a Chinese herb I-Tiao-Gung

The anti-oxidative and anti-inflammatory activities of two different species of traditional Chinese medicines that shared the same name have been studied. The extracts of Glycine radix have higher activities in free radical-scavenging activity determined with DPPH, reduction in hemoglobin-catalyzed lipid auto-oxidation and inhibition of the lipoxygenase (LOX) and cyclooxygenase (COX)-catalyzed arachidonate oxidation compared to the activities of extract of Flemingia. One of the significant bioactive constituents of Glycine radix has been isolated and identified as daidzein.     

Transmission and diversity of Schistosoma haematobium and S. bovis and their freshwater intermediate snail hosts Bulinus globosus and B. nasutus in the Zanzibar Archipelago,United Republic of Tanzania

BackgroundThe Zanzibar Archipelago (Pemba and Unguja islands) is targeted for the elimination of human urogenital schistosomiasis caused by infection with Schistosoma haematobium where the intermediate snail host is Bulinus globosus. Following multiple studies, it has remained unclear if B. nasutus (a snail species that occupies geographically distinct regions on the Archipelago) is involved in S. haematobium transmission on Zanzibar. Additionally, S. haematobium was thought to be the only Schistosoma species present on the Zanzibar Archipelago until the sympatric transmission of S. bovis, a parasite of ruminants, was recently identified. Here we re-assess the epidemiology of schistosomiasis on Pemba and Unguja together with the role and genetic diversity of the Bulinus spp. involved in transmission.Methodology/Principal findingsMalacological and parasitological surveys were conducted between 2016 and 2019. In total, 11,116 Bulinus spp. snails were collected from 65 of 112 freshwater bodies surveyed. Bulinus species identification were determined using mitochondrial cox1 sequences for a representative subset of collected Bulinus (n = 504) and together with archived museum specimens (n = 6), 433 B. globosus and 77 B. nasutus were identified. Phylogenetic analysis of cox1 haplotypes revealed three distinct populations of B. globosus, two with an overlapping distribution on Pemba and one on Unguja. For B. nasutus, only a single clade with matching haplotypes was observed across the islands and included reference sequences from Kenya. Schistosoma haematobium cercariae (n = 158) were identified from 12 infected B. globosus and one B. nasutus collected between 2016 and 2019 in Pemba, and cercariae originating from 69 Bulinus spp. archived in museum collections. Schistosoma bovis cercariae (n = 21) were identified from seven additional B. globosus collected between 2016 and 2019 in Pemba. By analysing a partial mitochondrial cox1 region and the nuclear ITS (1–5.8S-2) rDNA region of Schistosoma cercariae, we identified 18 S. haematobium and three S. bovis haplotypes representing populations associated with mainland Africa and the Indian Ocean Islands (Zanzibar, Madagascar, Mauritius and Mafia).Conclusions/SignificanceThe individual B. nasutus on Pemba infected with S. haematobium demonstrates that B. nasutus could also play a role in the local transmission of S. haematobium. We provide preliminary evidence that intraspecific variability of S. haematobium on Pemba may increase the transmission potential of S. haematobium locally due to the expanded intermediate host range, and that the presence of S. bovis complicates the environmental surveillance of schistosome infections.