Mutations of the tumor suppressor gene p53 are the most common genetic alterations observed in human cancer. Loss of wild-type p53 function impairs cell cycle arrest as well as repair mechanisms involved in response to DNA damage. Further, apoptotic pathways as induced by radio- or chemotherapy are also abrogated. Gene transfer of wild-type p53 was shown to reverse these deficiencies and to induce apoptosis in vitro and in preclinical in vivo tumor models. A phase I dose escalation study of a single intratumoral injection of a replication-defective adenoviral expression vector encoding wild-type p53 was carried out in patients with incurable non-small cell lung cancer. All patients enrolled had p53 protein overexpression as a marker of mutant p53 status in pretreatment tumor biopsies. Treatment was performed either by bronchoscopic intratumoral injection or by CT-guided percutaneous intratumoral injection of the vector solution. Fifteen patients were enrolled in two centers, and were treated at four different dose levels ranging from 10(7) to 10(10) PFU (7.5 x 10(9) to 7.5 x 10(12) particles). No clinically significant toxicity was observed. Successful transfer of wild-type p53 was achieved only with higher vector doses. Vector-specific wild-type p53 RNA sequences could be demonstrated in posttreatment biopsies of six patients. Transient local disease control by a single intratumoral injection of the vector solution was observed in four of those six successfully transduced patients. There was no evidence of clinical responses at untreated tumor sites. Wild-type p53 gene therapy by intratumoral injection of a replication-defective adenoviral expression vector is safe, feasible, and biologically effective in patients with advanced non-small cell lung cancer.

Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.Invasive aspergillosis (IA), primarily caused by the mold Aspergillus fumigatus, is a devastating disease in immunocompromised patients suffering from hematological malignancies or undergoing allogeneic hematopoietic stem cell transplantation (HSCT) 1 . The mortality rate of HSCT patients diagnosed with IA ranges from 60-90% 2 and the prognosis for long-term survival is extremely poor 3 . Recently, it was shown that HSCT patients with probable/proven IA had a delayed reconstitution of natural killer (NK) cells for more than a year post HSCT 4 . In addition, patients with severe IA were found to have a lower NK cell count compared to patients with well-controlled IA, suggesting that NK cells play a critical role in immunity to IA.NK cells comprise 5-15% of the peripheral blood mononuclear cells (PBMCs) in healthy individuals and belong to the innate immune system 5 . Upon activation, NK cells release immune regulatory cytokines to stimulate other immune cells and display cytotoxicity directed against tumor or virus-infected cells by granule release 5 . NK cells are defined as CD56 positive and CD3 negative cells and can be distinguished into CD3 − CD56 dim CD16 + and CD3 − CD56 bright CD16 − cells. While CD56 dim cells are more cytotoxic, CD56 bright cells produce high levels of cytokines such as IFNγ and TNFα 6 . The function of NK cells is induced by the interplay of inhibitory and activating receptors 7 , leading to cytotoxicity directed against tumors and virus-infected cells. Besides the recognition of these cells, NK cells also recognize other infectious pathogens, become activated, and as a response induce either lysis of these pathogens or trigger activation of other...

We observed a strong association between turtle exposure and Salmonella infections in this outbreak. Small turtles continue to be sold and pose a health risk, especially to children; many people remain unaware of the link between Salmonella infection and reptile contact.

Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C6 and long-chain C16-ceramides and azido-functionalized ceramide analogs against pathogenic Neisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C6-ceramide were active against Neisseria meningitidis and N. gonorrhoeae, whereas they were inactive against Escherichia coli and Staphylococcus aureus. Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω–azido-C6-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω–azido-C6-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenic Neisseriae.