Distinctive Immunomodulatory and Inflammatory Properties of the Escherichia coli Type II Heat-Labile Enterotoxin LT-IIa and Its B Pentamer following Intradermal Administration

The type I and type II heat-labile enterotoxins (LT-I and LT-II) are strong mucosal adjuvants when they are coadministered with soluble antigens. Nonetheless, data on the parenteral adjuvant activities of LT-II are still limited. Particularly, no previous study has evaluated the adjuvant effects and induced inflammatory reactions of LT-II holotoxins or their B pentameric subunits after delivery via the intradermal (i.d.) route to mice. In the present report, the adjuvant and local skin inflammatory effects of LT-IIa and its B subunit pentamer (LT-IIaB(5)) were determined. When coadministered with ovalbumin (OVA), LT-IIa and, to a lesser extent, LT-IIaB(5) exhibited serum IgG adjuvant effects. In addition, LT-IIa but not LT-IIaB(5) induced T cell-specific anti-OVA responses, particularly in respect to induction of antigen-specific cytotoxic CD8(+) T cell responses. LT-IIa and LT-IIaB(5) induced differential tissue permeability and local inflammatory reactions after i.d. injection. Of particular interest was the reduced or complete lack of local reactions, such as edema and tissue induration, in mice i.d. inoculated with LT-IIa and LT-IIaB(5), respectively, compared with mice immunized with LT-I. In conclusion, the present results show that LT-IIa and, to a lesser extent, LT-IIaB(5) exert adjuvant effects when they are delivered via the i.d. route. In addition, the low inflammatory effects of LT-IIa and LT-IIaB(5) in comparison to those of LT-I support the usefulness of LT-IIa and LT-IIaB(5) as parenterally delivered vaccine adjuvants.

Increase in S6K1 phosphorylation in human skeletal muscle following resistance exercise occurs mainly in type II muscle fibres

To investigate the in vivo effects of resistance exercise on translational control in human skeletal muscle, we determined the phosphorylation of AMP-activated kinase (AMPK), eukaryotic initiation factor 4E-binding protein (4E-BP1), p70/p85-S6 protein kinase (S6K1), and ribosomal S6 protein (S6). Furthermore, we investigated whether changes in the phosphorylation of S6K1 are muscle fiber type specific. Eight male subjects performed a single high-intensity resistance exercise session. Muscle biopsies were collected before and immediately after exercise and after 30 and 120 min of postexercise recovery. The phosphorylation statuses of AMPK, 4E-BP1, S6K1, and S6 were determined by Western blotting with phospho-specific and pan antibodies. To determine fiber type-specific changes in the phosphorylation status of S6K1, immunofluorescence microscopy was applied. AMPK phosphorylation was increased approximately threefold immediately after resistance exercise, whereas 4E-BP1 phosphorylation was reduced to 27 ± 6% of preexercise values. Phosphorylation of S6K1 at Thr421/Ser424 was increased 2- to 2.5-fold during recovery but did not induce a significant change in S6 phosphorylation. Phosphorylation of S6K1 was more pronounced in the type II vs. type I muscle fibers. Before exercise, phosphorylated S6K1 was predominantly located in the nuclei. After 2 h of postexercise recovery, phospho-S6K1 was primarily located in the cytosol of type II muscle fibers. We conclude that resistance exercise effectively increases the phosphorylation of S6K1 on Thr421/Ser424, which is not associated with a substantial increase in S6 phosphorylation in a fasted state.

Transcriptional regulation of the yghJ-pppA-yghG-gspCDEFGHIJKLM cluster, encoding the type II secretion pathway in Enterotoxigenic Escherichia coli

The gene cluster gspCDEFGHIJKLM codes for various structural components of the type II secretion pathway which is responsible for the secretion of heat-labile enterotoxin by enterotoxigenic Escherichia coli (ETEC). In this work, we used a variety of molecular approaches to elucidate the transcriptional organization of the ETEC type II secretion system and to unravel the mechanisms by which the expression of these genes is controlled. We showed that the gspCDEFGHIJKLM cluster and three other upstream genes, yghJ, pppA, and yghG, are cotranscribed and that a promoter located in the upstream region of yghJ plays a major role in the expression of this 14-gene transcriptional unit. Transcription of the yghJ promoter was repressed 168-fold upon a temperature downshift from 37°C to 22°C. This temperature-induced repression was mediated by the global regulatory proteins H-NS and StpA. Deletion mutagenesis showed that the promoter region encompassing positions −321 to +301 relative to the start site of transcription of yghJ was required for full repression. The yghJ promoter region is predicted to be highly curved and bound H-NS or StpA directly. The binding of H-NS or StpA blocked transcription initiation by inhibiting promoter open complex formation. Unraveling the mechanisms of regulation of type II secretion by ETEC enhances our understanding of the pathogenesis of ETEC and other pathogenic varieties of E. coli.

The type II secretion system and its ubiquitous lipoprotein substrate, SsIE are required for biofilm formation and virulence of enteropathogenic escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenic E. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease.

Are the preference and selection patterns of hermit crabs for gastropod shells species- or site-specific?

Experimental analyses of hermit crabs and their preferences for shells are essential to understand the intrinsic relationship of the crabs' dependence on shells, and may be useful to explain their shell use pattern in nature. The aim of this study was to evaluate the effect of crab species and site on the pattern of shell use, selection, and preference in the south-western Atlantic hermit crabs Pagurus brevidactylus and Pagurus criniticornis, comparing sympatric and allopatric populations. Differently from the traditional approach to evaluate shell preference by simply determining the shell selection pattern (i.e., the number of shells of each type selected), preference was defined (according to [Liszka, D., Underwood, AJ., 1990. An experimental design to determine preferences for gastropod shells by a hermit-crab. J. Exp. Mar. Biol. Ecol., 137(1), 47-62]) by the comparison of the number of crabs changing for a particular shell type when three options were given (Cerithium atratum, Morula nodulosa, and Tegula viridula) with the number of crabs changing for this same type when only this type was offered. The effect of crab species was tested at Cabelo Gordo Beach, where P. brevidacrylus was found occupying shells of C. atratum, M. nodulosa, and T viridula in similar frequencies, whereas P. criniticornis occupied predominantly shells of C atratum. In laboratory experiments the selection patterns of the two hermit-crab species for these three gastropods were different, with P criniticornis selecting mainly shells of C atratum, and R brevidactylus selecting more shells of M. nodulosa. The shell preference was also dependent on crab species, with P. criniticornis showing a clear preference for shells of C atratum, whereas P. brevidactylus did not show a preference for any of the tested shells. The effect of site was tested for the two species comparing data from Cabelo Gordo to Preta (P brevidactylus) and Araca beaches (P. criniticornis). The pattern of shell use, selection, and preference was demonstrated to be dependent on site only for P. brevidactylus. The results also showed that the shell use pattern of P criniticornis can be explained by its preference at both sites, whereas for P. brevidactylus it occurred only at Cabelo Gordo, where the absence of preference was correlated with the similar use of the three gastropod species studied. Finally, the results showed that the shell selection pattern cannot be considered as a measure of shell preference, since it overestimates crab selectivity. (C) 2009 Elsevier B.V. All rights reserved.

Exfoliative cytology of the oral mucosa in type II diabetic patients: morphology and cytomorphometry

Background: In recent years, important advances have occurred in the determination of diagnostic criteria for the disease diabetes mellitus and in new strategies for its treatment. The purpose of this research was to develop a new method for diabetes diagnosis by microscopic and cytomorphometric analyses of the oral epithelium. Methods: the smears were obtained from three distinct oral sites: buccal mucosa (cheek), tongue dorsum, and floor of the mouth in 10 control individuals and 10 type II diabetic patients. The oral smears were stained with Papanicolaou EA-36 solution. The nuclear (NA) and cytoplasmic (CA) areas were evaluated from 50 integral cells predominant in each oral site by the use of the KS 300(TM) image analysis system (Carl Zeiss, Germany), by which the cytoplasmic/nuclear ratio (C/N) was calculated. Results: the results showed that: (i) the epithelial cells of the diabetic group exhibited figures of binucleation and occasional karyorrhexis in all layers; (ii) the NA was markedly higher (P<0.05) in the diabetic group; (iii) the CA did not exhibit a statistically significant difference (P>0.05) between these two groups; and (iv) the C/N mean was 37.4% lower in the type II diabetic group. Conclusions: These results associated with clinical observations suggest that diabetes mellitus can produce alterations in oral epithelial cells, detectable by microscopy and cytomorphometry, which can be used in the diagnosis of this disease.

Are the preference and selection patterns of hermit crabs for gastropod shells species- or site-specific?

Experimental analyses of hermit crabs and their preferences for shells are essential to understand the intrinsic relationship of the crabs` dependence on shells, and may be useful to explain their shell use pattern in nature. The aim of this study was to evaluate the effect of crab species and site on the pattern of shell use, selection, and preference in the south-western Atlantic hermit crabs Pagurus brevidactylus and Pagurus criniticornis, comparing sympatric and allopatric populations. Differently from the traditional approach to evaluate shell preference by simply determining the shell selection pattern (i.e., the number of shells of each type selected), preference was defined (according to [Liszka, D., Underwood, AJ., 1990. An experimental design to determine preferences for gastropod shells by a hermit-crab. J. Exp. Mar. Biol. Ecol., 137(1), 47-62]) by the comparison of the number of crabs changing for a particular shell type when three options were given (Cerithium atratum, Morula nodulosa, and Tegula viridula) with the number of crabs changing for this same type when only this type was offered. The effect of crab species was tested at Cabelo Gordo Beach, where P. brevidacrylus was found occupying shells of C. atratum, M. nodulosa, and T viridula in similar frequencies, whereas P. criniticornis occupied predominantly shells of C atratum. In laboratory experiments the selection patterns of the two hermit-crab species for these three gastropods were different, with P criniticornis selecting mainly shells of C atratum, and R brevidactylus selecting more shells of M. nodulosa. The shell preference was also dependent on crab species, with P. criniticornis showing a clear preference for shells of C atratum, whereas P. brevidactylus did not show a preference for any of the tested shells. The effect of site was tested for the two species comparing data from Cabelo Gordo to Preta (P brevidactylus) and Araca beaches (P. criniticornis). The pattern of shell use, selection, and preference was demonstrated to be dependent on site only for P. brevidactylus. The results also showed that the shell use pattern of P criniticornis can be explained by its preference at both sites, whereas for P. brevidactylus it occurred only at Cabelo Gordo, where the absence of preference was correlated with the similar use of the three gastropod species studied. Finally, the results showed that the shell selection pattern cannot be considered as a measure of shell preference, since it overestimates crab selectivity. (C) 2009 Elsevier B.V. All rights reserved.

Mechanism and Efficiency of Cell Death of Type II Photosensitizers: Effect of Zinc Chelation

A series of meso-substituted tetra-cationic porphyrins, which have methyl and octyl substituents, was studied in order to understand the effect of zinc chelation and photosensitizer subcellular localization in the mechanism of cell death. Zinc chelation does not change the photophysical properties of the photosensitizers (all molecules studied are type II photosensitizers) but affects considerably the interaction of the porphyrins with membranes, reducing mitochondrial accumulation. The total amount of intracellular reactive species induced by treating cells with photosensitizer and light is similar for zinc-chelated and free-base porphyrins that have the same alkyl substituent. Zinc-chelated porphyrins, which are poorly accumulated in mitochondria, show higher efficiency of cell death with features of apoptosis (higher MTT response compared with trypan blue staining, specific acridine orange/ethidium bromide staining, loss of mitochondrial transmembrane potential, stronger cytochrome c release and larger sub-G1 cell population), whereas nonchelated porphyrins, which are considerably more concentrated in mitochondria, triggered mainly necrotic cell death. We hypothesized that zinc-chelation protects the photoinduced properties of the porphyrins in the mitochondrial environment.

Isolated GH deficiency type II: knockdown of the harmful Delta3GH recovers wt-GH secretion in rat tumor pituitary cells

Isolated GH deficiency type II (IGHD II) is the autosomal dominant form of GHD. In the majority of the cases, this disorder is due to specific GH-1 gene mutations that lead to mRNA missplicing and subsequent loss of exon 3 sequences. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH (Delta3GH) isoform that reduces the accumulation and secretion of wild-type-GH. At present, patients suffering from this type of disease are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the Delta3GH mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. We developed a strategy involving Delta3GH isoform knockdown mediated by expression of a microRNA-30-adapted short hairpin RNA (shRNA) specifically targeting the Delta3GH mRNA of human (shRNAmir-Delta3). Rat pituitary tumor GC cells expressing Delta3GH upon doxycycline induction were transduced with shRNAmir-Delta3 lentiviral vectors, which significantly reduced Delta3GH protein levels and improved human wild-type-GH secretion in comparison with a shRNAmir targeting a scrambled sequence. No toxicity due to shRNAmir expression could be observed in cell proliferation assays. Confocal microscopy strongly suggested that shRNAmir-Delta3 enabled the recovery of GH granule storage and secretory capacity. These viral vectors have shown their ability to stably integrate, express shRNAmir, and rescue IGHD II phenotype in rat pituitary tumor GC cells, a methodology that opens new perspectives for the development of gene therapy to treat IGHD patients.

Gonadotropin-releasing hormone type II antagonist induces apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells in vitro and in vivo

Triple-negative breast cancer does not express estrogen and progesterone receptors, and no overexpression/amplification of the HER2-neu gene occurs. Therefore, this subtype of breast cancer lacks the benefits of specific therapies that target these receptors. Today chemotherapy is the only systematic therapy for patients with triple-negative breast cancer. About 50% to 64% of human breast cancers express receptors for gonadotropin-releasing hormone (GnRH), which might be used as a target. New targeted therapies are warranted. Recently, we showed that antagonists of gonadotropin-releasing hormone type II (GnRH-II) induce apoptosis in human endometrial and ovarian cancer cells in vitro and in vivo. This was mediated through activation of stress-induced mitogen-activated protein kinases (MAPKs) p38 and c-Jun N-terminal kinase (JNK), followed by activation of proapoptotic protein Bax, loss of mitochondrial membrane potential, and activation of caspase-3. In the present study, we analyzed whether GnRH-II antagonists induce apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells that express GnRH receptors. In addition, we ascertained whether knockdown of GnRH-I receptor expression affects GnRH-II antagonist-induced apoptosis and apoptotic signaling.

Sperm-binding fibronectin type II-module proteins are genetically linked and functionally related

Fibronectin type II (Fn2) module-containing proteins in the male genital tract are characterized by different numbers of Fn2 modules. Predominantly two classes exist which are distinct by having either two or four Fn2 modules. Minor variants with three Fn2 modules were also found in the human and the porcine epididymis. To reveal their relationship, mRNAs and proteins of representatives of these classes were studied in human, in Sus scrofa, and in rodents. Adult boars expressed members of both classes, i.e. ELSPBP1 and pB1, in subsequent regions of the epididymis, and both were under androgenic control. Human and rodent epididymides, on the other hand, alternatively contained only representatives of one of these two classes, i.e. ELSPBP1 in the human and two different pB1-related counterparts in rodents. ELSPBP1 and pB1-related genomic sequences were closely linked in chromosomal regions HSA 19q and SSC 6 q11-q21; conserved synteny between these regions is well established. On the other hand, in a syntenic region on mouse chromosome 7, ELSPBP1-related sequences were lacking. Tight binding to the sperm membrane via a choline-mediated mechanism was a common feature of the two classes of Fn2-module proteins, suggesting related function(s). However, differences in their regionalized expression patterns along the male genital tract as well as in association sites on the sperm surface suggested a species-specific sequential order in sperm binding.

[Drug therapy of type-II diabetes: tablets, insulin or a combination of these]

Optimal therapy of diabetes has to be based on the known pathophysiology of metabolic disturbances and should eventually alleviate reduced secretion of insulin as well as reduce the usually present resistance to insulin in order to normalize the average blood glucose levels. In less than 30% of patients with type-II diabetes, dietetic measures combined with increased physical activity alone, are sufficient for metabolic control, thus increasing the importance of pharmacologic treatment immensely. Biguanides are the therapeutic choice in patients with massive overweight, because they usually do not induce weight gain; however, specific contraindications (renal failure in particular) have to be taken into consideration. The effect of blood glucose lowering by biguanides is not due to increased secretion of insulin, thus neither hypoglycemias nor hyperinsulinism are induced or increased, respectively. Patients with normal or slightly increased body weight should profit best from sulfonylureas that stimulate insulin production. Combinations of sulfonylurea and biguanides or of insulin and oral antidiabetics or insulin alone have to be taken into account when monotherapy with oral antidiabetics is too inefficient; however, clear and generally accepted guidelines for correct indications of these therapeutic modalities are lacking. Particularly in long-lasting diabetes and for patients with distinct overweight an adequate therapeutic success is often not obtained with the currently available therapeutic means. Possibly, future developments will provide new therapeutic ways with drugs that increase insulin sensitivity or reduce gluconeogenesis.

Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes

Fas/CD95-induced apoptosis of hepatocytes in vivo proceeds through the so-called type II pathway, requiring the proapoptotic BH3-only Bcl-2 family member Bid for mitochondrial death signaling. Consequently, Bid-deficient mice are protected from anti-Fas antibody injection induced fatal hepatitis. We report the unexpected finding that freshly isolated mouse hepatocytes, cultured on collagen or Matrigel, become independent of Bid for Fas-induced apoptosis, thereby switching death signaling from type II to type I. In such in vitro cultures, Fas ligand (FasL) activates caspase-3 without Bid cleavage, Bax/Bak activation or cytochrome c release, and neither Bid ablation nor Bcl-2 overexpression is protective. The type II to type I switch depends on extracellular matrix adhesion, as primary hepatocytes in suspension die in a Bid-dependent manner. Moreover, the switch is specific for FasL-induced apoptosis as collagen-plated Bid-deficient hepatocytes are protected from tumor necrosis factor alpha/actinomycin D (TNFalpha/ActD)-induced apoptosis. Conclusion: Our data suggest a selective crosstalk between extracellular matrix and Fas-mediated signaling that favors mitochondria-independent type I apoptosis induction.

Targeted gene transfer of hepatocyte growth factor to alveolar type II epithelial cells reduces lung fibrosis in rats

Inefficient alveolar wound repair contributes to the development of pulmonary fibrosis. Hepatocyte growth factor (HGF) is a potent growth factor for alveolar type II epithelial cells (AECII) and may improve repair and reduce fibrosis. We studied whether targeted gene transfer of HGF specifically to AECII improves lung fibrosis in bleomycin-induced lung fibrosis. A plasmid encoding human HGF expressed from the human surfactant protein C promoter (pSpC-hHGF) was designed, and extracorporeal electroporation-mediated gene transfer of HGF specifically to AECII was performed 7 days after bleomycin-induced lung injury in the rat. Animals were killed 7 days after hHGF gene transfer. Electroporation-mediated HGF gene transfer resulted in HGF expression specifically in AECII at biologically relevant levels. HGF gene transfer reduced pulmonary fibrosis as assessed by histology, hydroxyproline determination, and design-based stereology compared with controls. Our results indicate that the antifibrotic effect of HGF is due in part to a reduction of transforming growth factor-β(1), modulation of the epithelial-mesenchymal transition, and reduction of extravascular fibrin deposition. We conclude that targeted HGF gene transfer specifically to AECII decreases bleomycin-induced lung fibrosis and may therefore represent a novel cell-specific gene transfer technology to treat pulmonary fibrosis.

Serotyping of Toxoplasma gondii in cats (Felis domesticus) reveals predominance of type II infections in Germany.

BACKGROUND Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany. METHODOLOGY To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7). FINDINGS Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III. CONCLUSIONS Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.