Homeostasis of peripheral FoxP3+ CD4+ regulatory T cells in patients with early and late stage breast cancer

FoxP3 ⁺ CD4 ⁺ regulatory T cells (Tregs) are important mediators of peripheral immune tolerance, acting via multiple mechanisms to suppress cellular immunity including antitumor responses. Although therapeutic strategies have been proposed to deplete Tregs in patients with breast cancer and other malignancies, dynamic changes in the Treg compartment as a function of stage and treatment of breast cancer remain poorly understood. Here, we evaluated peripheral blood CD4⁺ T cells and FoxP3⁺ CD4⁺ T cells from 45 patients with early or late stage breast cancer and compared percentages, absolute counts, and Treg function to those from healthy volunteers (HV) of comparable age. Patients having completed adjuvant chemotherapy and patients with metastatic cancer exhibited significantly lower absolute CD4 counts and significantly higher percentages of FoxP3⁺ CD4⁺ T cells. In contrast, the absolute counts of circulating FoxP3⁺ CD4⁺ T cells did not differ significantly among early stage patients, late stage patients, or HV. Functionally, FoxP3⁺ CD4⁺ T cells from all donor groups similarly expressed CTLA-4 and failed to secrete IFN-γ in response to stimulation. Thus, although Tregs comprise an increased percentage of circulating CD4⁺ T cells in patients with metastatic breast cancer and patients in remission after completing the adjuvant chemotherapy, the systemic Treg pool, as measured by absolute counts, appears relatively constant regardless of disease stage or treatment status. Total CD4⁺ T cell counts are not constant, however, suggesting that homeostatic mechanisms, or susceptibility to cytotoxic or malignant insults, fundamentally differ for regulatory and non-regulatory CD4⁺ T cells.     

A Cytohesin Homolog in Dictyostelium Amoebae

Background

Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration.

Principal Findings

Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG⁻ cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced.

Significance

The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.     

Reversion of a truncated gene for ampicillin resistance by genetic rearrangements in Escherichia coli K12

Summary The composite transposon Tn2672 is a derivative of the Tn3-related transposon Tn902 whose bla gene providing ampicillin resistance had been inactivated by the insertion of the IS1-flanked multiple drug resistance transposon Tn2671. Most ampicillin resistant revertants of Tn2672 are due to precise excision of Tn2671. However, a rare Bla⁺ revertant which still retains all the previously acquired drug resistance markers was isolated. On this revertant, the 5 part of the split bla gene on Tn2672 has converted to an intact, active bla gene, and the entire Tn902 is structurally restored. In contrast, the adjacent IS1b element belonging to Tn2671 has its terminal 142 base pairs deleted. Despite of this rearragement, the split 3 part of bla and its adjacent sequences have remained unchanged. Models are presented to explain the observed DNA rearrangements, and their similarity with gene conversion events is discussed.     

[3H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Brain astroglial cells, whether from a bulk isolated preparation or in culture, have been shown to take up serotonin actively. [3H]imipramine has been proposed as a specific label for serotonin uptake sites in brain. We therefore studied the binding of [3H]imipramine to C6 astroglial cells in culture to determine if some of the binding of this radioligand in brain homogenates is actually to serotonin transporting sites on glia. [3H]Imipramine binds saturably (Bmax = 202 fmol/mg protein) and with high affinity (KD = 1.72 nM) to C6 cells. This binding is competitively inhibited by other tricyclic antidepressants. The C6 cells actively transport [3H]serotonin with a Km of 2 microM and a Vmax of 1080 fmol/10(6) cells/min. However, the pharmacological profile for inhibition of serotonin uptake does not correlate with the pharmacological profile for inhibition of [3H]imipramine binding. These results suggest that the binding of [3H]imipramine to astroglial cells is not related to their capacity for active uptake of serotonin. Further, in brain homogenates, some of the binding of [3H]imipramine may not be to neuronal uptake sites but rather may be to sites on astroglial cells.     

Growth of Bacillus coagulans in Chemically Defined Media

A nutritional study was made of five strains of Bacillus coagulans obtained from various culture collections. These five strains were descendants of two original isolates; three had been derived from one parent culture in years past and the other two were transfers from another parent culture. Therefore, the five cultures should have represented two distinct groups of genetically identical cultures. Three of the strains obtained from one culture collection had become methyl red-negative and sorbitol-negative and had gained abilities to hydrolyze gelatin and ferment arabinose. Nutritional requirements of the five cultures, determined at 37, 45, and 55 C, differed considerably among strains; however, thiamine and biotin were required by all cultures at all temperatures. Aspartic acid was stimulatory at 37 C and was required at 45 C; folic acid, basic amino acids, and certain other nutrilites were required at 55 C. Adenine supplementation was necessary for two strains at 55 C to prevent autolysis; this phenomenon is discussed. The response of these organisms to both serine and the basic amino acids at the three growth temperatures seems especially significant. The media devised for the growth of the five strains of B. coagulans used in this study permit excellent growth at three incubation temperatures.     

Biochemical Characterisation and Localization in Brain of a Human Brain-Leucocyte Membrane Glycoprotein Recognised by a Monoclonal Antibody

Abstract: The F10-44-2 monoclonal antibody was originally shown to interact with a determinant found predominantly in human brain and leucocytes. In this study we demonstrate by quantitative absorption analysis with homogenates of the head of the caudate nucleus, putamen, thalamus, cerebral grey matter, cerebral white matter, corpus callosum and cerebellar folia that the determinant is restricted to the white matter of the CNS. Immunofluorescence studies on frozen sections of the above brain subregions confirm the absorption analyses, showing staining only of white matter. In addition, and unexpectedly, we found very bright staining around blood vessels, particularly in the cerebellum. Biochemical studies established that the molecule in white matter bearing the F10-44-2 determinants is a sialylated membrane glycoprotein with an apparent molecular weight of 90,000, which is similar to but slightly smaller than the T lymphocyte form of the antigen. Developmental studies comparing 16-week foetal and adult cerebrum showed a fivefold increase in F10-44-2 antigen content. Thus, in the human CNS, the F10-44-2 antigen is a medium-sized glycoprotein which is restricted to white matter and shows a marked increase in concentration during development. No such molecule has been described previously.     

Microautoradiographic detection of CO2 fixation in lenticel chlorenchyma of youngFraxinus excelsior L. stems in early spring

Microautoradiography indicated that 1-year-oldFraxinus excelsior L. stem chlorenchyma assimilated external¹⁴CO₂ in mid-April, when buds were swollen, but before bud-break. The lenticel regions showed the highest amount of radioactively labeled assimilates. Labeled assimilates declined in the tangential direction with increasing distance from lenticels, suggesting that¹⁴CO₂ entered the stem through the open intercellular spaces of lenticels. In the radial direction, the amount of radioactively labeled assimilates did not constantly decline with growing distance from the lenticel entrance. It was high in all lenticel phelloderm cells, which had high chlorophyll autofluorescence and very small starch grains, highest in the adjacent 4–6 rows of chlorenchyma, which had larger starch grains that increased in size towards the interior rows, and much lower in the inner cortex chlorenchyma, which had large starch grains. We suggest that the main function of the lenticel chlorenchyma (lenticel phelloderm plus 4–6 rows of adjacent cortex chlorenchyma) is the refixation of respiratory CO₂ which could easily leave the stem intercellular spaces, rather than the fixation of external CO₂. The lenticel chlorenchyma could reduce the loss of respiratory CO₂ by its photosynthetic activity.     

Upregulation of (+)-7-Hydroxy-N,N-di-n-[3H]Propyl-2-Aminotetralin Binding Following Intracerebroventricular Administration of a Nitric Oxide Generator

Nitric oxide modulation of dopamine D₂ and D₃ receptor binding was examined using [¹²⁵I]epidepride (D₂) and (+)7-hydroxy-N,N-di-n-[³H]propyl-2-aminotetralin([³H](+)-7-OH-DPAT, D₃). Nitric oxide, generated by i.c.v. injection of S-nitroso-N-acetyl-penicillamine (SNAP; 5 g or 10 g), significantly increased the density of [³H](+)-7-OH-DPAT binding sites (39% and 134%, respectively) in the striatum 24 hours post-injection in the absence of Gpp(NH)p, representing an upregulation of either D₃, receptors or high affinity D₂ receptors. In the presence of 10 M Gpp(NH)p, D₃ receptor upregulation was maintained in both the 5 g (increased 35%) and 10 g SNAP (increased 44%) groups. [³H](+)-7-OH-DPAT binding was reduced in both striatum and nucleus accumbens in the presence of 10 M Gpp(NH)p compared to binding in the absence of Gpp(NH)p, suggesting an upregulation of D₃ receptors. Administration of SNAP did not alter total specific [¹²⁵I]epidepride binding in either brain region. These data suggest that; 1) D₃ receptor density is modified following nitric oxide generation, and 2) the density of high affinity D₂ receptors identified by [³H](+)-7-OH-DPAT increases in the striatum, but decreases in the nucleus accumbens.     

Fine structure of the fat body and nephrocytes in the life-stages ofDermacentor variabilis

The fine structure of the fat body and associated nephrocytes of the American dog tick,Dermacentor variabilis (Say), was described in unfed larvae, unfed nymphs, and in unfed and fed adults of both sexes. The fat body consisted of one type of cell, the trophocyte. Morphological changes that occured in the trophocytes of both sexes were dependent on feeding. The ultrastructure of feeding male trophocytes was distinct from trophocytes of feeding females. In the feeding female, the trophocyte developed an ultrastructure characteristic of cells that produce secretory proteins. A type of scalariform cell junction was found associated with rough endoplasmic reticulum of the trophocytes. Nephrocytes were closely associated with trophocytes but were not part of the fat body. Nephrocyte ultrastructure was unaltered throughout the life-stages we examined, except at the end of oviposition. Organelles in the nephrocytes were not randomly distributed, but were found in distinct regions of the cytoplasm. Slit diaphragms at the surface of the nephrocytes were extracellular specializations that had a periodic ultrastructure.     

THE CIRCADIAN RHYTHM OF BIOLUMINESCENCE IN PYROCYSTIS IS NOT DUE TO DIFFERENCES IN THE AMOUNT OF LUCIFERASE: A COMPARATIVE STUDY OF THREE BIOLUMINESCENT MARINE DINOFLAGELLATES

The biochemistry and circadian regulation of luminescence in two Pyrocystis species, P. lunula Hulburt and P. noctiluca Murray et Haeckel, were compared with a well-studied species, Gonyaulax polyedra Stein. All exhibit circadian rhythms and all have similar luciferins and luciferases. However, the Pyrocystis species lack a second protein involved in the reaction in Gonyaulax , the luciferin (substrate) binding protein, which sequesters the luciferin at the cytoplasmic pH and releases it upon acidification, thus controlling the characteristic flashing, which is similar in the three species. More striking is the difference in the circadian regulation of luminescence, which in Gonyaulax involves the daily synthesis and destruction of the two proteins, along with the luminous organelles (scintillons) from which light is emitted, and which are present in all species. In the Pyrocystis species, the amount of luciferase is the same in extracts made during the day and night phases; its circadian regulation in vivo may be attributed to a change in its localization from day to night phase.