In vivo expansion of naïve CD4+ CD25(high) FOXP3+ regulatory T cells in patients with colorectal carcinoma after IL-2 administration

Regulatory T cells (T_reg cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of T_reg cells was established. In IL-2 treated cancer patients a further T_reg-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional T_reg cells of a naïve phenotype - as determined by CCR7 and CD45RA expression - are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve T_reg-cell pool. Higher frequencies of T-cell receptor excision circles in naïve T_reg cells indicate IL-2 dependent thymic generation of naïve T_reg cells as a mechanism leading to increased frequencies of T_reg cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine T_reg cells after IL-2 administration. These results point to a more complex regulation of T_reg cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve T_reg cells.     

Membrankomplexe dictyosomaler Herkunft als „Matrizen“ für die extraplasmatische Synthese und Orientierung von Mikrofibrillen

Zusammenfassung Es wird ein neu gefundener Typ von Golgi-Vesikeln (F-Vesikeln), die während der Bildung der gemusterten Sekundärwand vonMicrasterias denticulata auftreten, eingehender beschrieben. Es wird gezeigt, daß speziell differenzierte Membranen dieser Vesikel wahrscheinlich als Matrizen für die Bildung der Sekundärwand-Mikrofibrillen fungieren.F-Vesikel entstehen in einem Prozeß der Membranreifung (Matrizen-Bildung) am distalen Pol der Dictyosomen. Von dort werden diese Vesikel, die globuläre Untereinheiten enthalten, welche reihenweise in der Vesikelmembran angeordnet sind, zur Plasmamembran transportiert (Matrizen-Transfer). Dort findet eine Inkorporation und Fusion der F-Vesikelmembran mit der Plasmamembran statt (Matrizen-Inkorporation). Diese inkorporierten und ausgebreiteten Membranareale scheinen bei der Produktion der Reihen von Mikrofibrillen von besonderer Bedeutung zu sein (Matrizen-Realisation).Die elektronenmikroskopischen Ergebnisse beiMicrasterias wurden mit jenen über die Schalenbildung bei Chrysophyceen (Pleurochrysis) verglichen. Dabei wurde eine überraschende Übereinstimmung bei der Wandbildung dieser beiden Organismen festgestellt: In beiden Fällen scheint es, daß Matrizen für die Bildung der Mikrofibrillen an den Membranen von Golgi-Vesikeln gebildet werden. BeiPleurochrysis erfolgt die Bildung der Wandelemente schon innerhalb der Golgi-Zisternen, während beiMicrasterias die Zelluloseproduktion verzögert ist (time gap) und erst nach Inkorporation der Vesikelmembran in die Plasmamembran beginnt.

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Dictyosome-Derived membrane complexes as templates for the extraplasmatic synthesis and orientation of microfibrils

Summary A newly found type of Golgi vesicles (F-vesicles), occuring during the formation of the highly patterned secondary wall ofMicrasterias denticulata is described in more detail. It is shown that specifically differentiated membranes of these vesicles are probably functional as templates for the formation of the secondary wall inMicrasterias. F-vesicles are formed in a process of membrane maturation (template formation) on the distal pole of the dictyosomes. From there these vesicles, carring globular subunits orientated in rows on the vesicle membrane, are transported to the plasma membrane (template transfer). Here an incorporation and fusion of the F-vesicle membrane with the plasma membrane occurs (template incorporation). These incorporated and exposed membrane areas seem to be involved in the production of the patterned arrays of microfibrils (template realization).The electron microscopic results inMicrasterias are compared with those described for scale formation in the chrysophycean algaPleurochrysis. A surprising relationship between the types of wall formation in these two organisms could be demonstrated: in both cases templates for the production of microfibrils seem to be formed on membranes of Golgi cisternae. InPleurochrysis the formation of wall elements takes place already within the Golgi cisterna, while inMicrasterias cellulose production is delayed (time gap) and begins just after the incorporation of the vesicle membrane into the plasma membrane.

     

How sex and age affect immune responses,susceptibility to infections,and response to vaccination

Do men die young and sick, or do women live long and healthy? By trying to explain the sexual dimorphism in life expectancy, both biological and environmental aspects are presently being addressed. Besides age-related changes, both the immune and the endocrine system exhibit significant sex-specific differences. This review deals with the aging immune system and its interplay with sex steroid hormones. Together, they impact on the etiopathology of many infectious diseases, which are still the major causes of morbidity and mortality in people at old age. Among men, susceptibilities toward many infectious diseases and the corresponding mortality rates are higher. Responses to various types of vaccination are often higher among women thereby also mounting stronger humoral responses. Women appear immune-privileged. The major sex steroid hormones exhibit opposing effects on cells of both the adaptive and the innate immune system: estradiol being mainly enhancing, testosterone by and large suppressive. However, levels of sex hormones change with age. At menopause transition, dropping estradiol potentially enhances immunosenescence effects posing postmenopausal women at additional, yet specific risks. Conclusively during aging, interventions, which distinctively consider the changing level of individual hormones, shall provide potent options in maintaining optimal immune functions.     

Cupredoxin-cancer interrelationship: azurin binding with EphB2, interference in EphB2 tyrosine phosphorylation, and inhibition of cancer growth

Azurin is a member of a family of metalloproteins called cupredoxins. Although previously thought to be involved in electron transfer, azurin has recently been shown to preferentially enter cancer cells than normal cells and induce apoptosis in such cells. Azurin also demonstrates structural similarity to a ligand known as ephrinB2, which binds its cognate receptor tyrosine kinase EphB2 to initiate cell signaling. Eph/ephrin signaling is known to be involved in cancer progression. We now demonstrate that azurin binds to the EphB2-Fc receptor with high affinity. We have localized a C-terminal domain of azurin (Azu 96-113) that exhibits structural similarity to ephrinB2 at the G-H loop region known to be involved in receptor binding. A synthetic peptide (Azu 96-113) as well as a GST fusion derivative GST-Azu 88-113 interferes with the growth of various human cancer cells. In a prostate cancer cell line DU145 lacking functional EphB2, azurin or its GST-fusion derivatives had little cytotoxic effect. However, in DU145 cells expressing functional EphB2, azurin and GST-Azu 88-113 demonstrated significant cytotoxicity, whereas ephrinB2 promoted cell growth. Azurin inhibited the ephrinB2-mediated autophosphorlyation of the EphB2 tyrosine residue, thus interfering in upstream cell signaling and contributing to cancer cell growth inhibition.     

Proteomics analysis of Thermoplasma acidophilum with a focus on protein complexes

Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known.     

Biological Relevance of Natural α-Toxin Fragments from Staphylococcus aureus

Serine proteases represent an essential part of cellular homeostasis by generating biologically active peptides. In bacteria, proteolysis serves two different roles: a major housekeeping function and the destruction of foreign or target cell proteins, thereby promoting bacterial invasion. In the process, other virulence factors such as exotoxins become affected. In Staphylococcus aureus culture supernatant, the pore-forming α-toxin is cleaved by the coexpressed V8 protease and aureolysin. The oligomerizing and pore-forming abilities of five such spontaneously occurring N- and C-terminal α-toxin fragments were studied. ³H-marked α-toxin fragments bound to rabbit erythrocyte membranes but only fragments with intact C termini, missing 8, 12 and 71 amino acids from their N-terminal, formed stable oligomers. All isolated fragments induced intoxication of mouse adrenocortical Y1 cells in vitro, though the nature of membrane damage for a fragment, degraded at its C terminus, remained obscure. Only one fragment, missing the first eight N-terminal amino acids, induced irreversible intoxication of Y1 cells in the same manner as the intact toxin. Four of the isolated fragments caused swelling, indicating altered channel formation. Fragments missing 12 and 71 amino acids from the N terminus occupied the same binding sites on Y1 cell membranes, though they inhibited membrane damage caused by intact toxin. In conclusion, N-terminal deletions up to 71 amino acids are tolerated, though the kinetics of channel formation and the channel’s properties are altered. In contrast, digestion at the C terminus results in nonfunctional species.