Bimodal ex vivo expansion of T cells from patients with head and neck squamous cell carcinoma: a prerequisite for adoptive cell transfer

Background aimsAdoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies.MethodsIn a two-step expansion process, initially TIL bulk cultures were established from primary and recurrent HNSCC tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics during culture. Interferon (INF)-γ detection by Elispot and Cr⁵¹ release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion.ResultsTIL bulk cultures were expanded in 80% of the patients included, showing tumor specificity in 60% of the patients. Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8⁺ and CD4⁺ subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded cultures, ranging from approximately 10 to 30 T-cell clonotypes. TIL from large-scale rapid expansions maintained functional capacity, and contained tumor-specific T cells.ConclusionThe procedure is feasible for expansion of TIL from HNSCC, ensuring clinically relevant expansion folds within 7 weeks. The cell culture kinetics and phenotypes of the TIL resemble previously published results on TIL from melanoma, setting the stage for clinical testing of this promising treatment strategy for patients with HNSCC.     

Detection of <Emphasis Type="Italic">Legionella</Emphasis> by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment

Background

Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool for risk assessment.

Results

In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6*10⁵GU/L while L. pneumophila were detected in a range from LOQ to 6.8*10⁵ GU/L. By culturing, the legionellae were detected in the range from below detection limit (> 10 CFU/L) to 1.6*10⁶ CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively poor (r² = 0.31 for culture and Legionella spp. assay, r² = 0.20 for culture and L. pneumophila assay).

Conclusion

Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc is dubious. However, the rapid detection by qPCR of high concentrations of Legionella - especially Legionella pneumophila - is valuable as an indicator of risk, although it may be false positive compared to culture results. On the other hand, the detection of a low number of bacteria by qPCR is a strong indication for the absence of risk.

     

No costs on freeze tolerance in genetically copper adapted earthworm populations (Dendrobaena octaedra)

For nearly three centuries the area around Gusum, in south-east Sweden, has been highly polluted with copper. An earlier study in this area showed that populations of the freeze-tolerant earthworm Dendrobaena octaedra were genetically adapted to copper. Apparently, no life-history costs to reproduction or growth were imposed by this adaptation. In the present paper we therefore investigated how laboratory raised F1-generations of these populations coped when exposed to increased copper concentrations in the soil and to sub-zero temperatures. We found that D. octaedra from polluted sites accumulated the same amount of copper as reference worms. Furthermore, earthworms from polluted sites survived equally to reference worms when exposed to freezing temperatures (-8 or -12°C). However, when simultaneously exposed to the lowest temperature and copper, the worms from polluted sites survived significantly better than reference worms. The overall conclusion of this study is that worms from polluted sites seem to be better at handling copper and accrue no costs in terms of reduced cold tolerance in connection to genetic adaptation in these populations.     

Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum

The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the use of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially expressed proteins, such as subunits of the succinate dehydrogenase complex and other TCA cycle enzymes that are usually found in the cytosol, thus hinting at a possible association to the membrane in response to nitrogen deficiency. We propose a redox sensing mechanism that can influence the membrane subproteome in response to nitrogen availability.     

In depth study of a new highly efficient raw starch hydrolyzing α-amylase from Rhizomucor sp

A new α-amylase from Rhizomucor sp. (RA) was studied in detail due to its very efficient hydrolysis of raw starch granules at low temperature (32 °C). RA contains a starch binding domain (SBD) connected to the core amylase catalytic domain by a O-glycosylated linker. The mode of degradation of native maize starch granules and, in particular, the changes in the starch structure during the hydrolysis, was monitored for hydrolysis of raw starch at concentrations varying between 0.1 and 31%. RA was compared to porcine pancreatic α-amylase (PPA), which has been widely studied either on resistant starch or as a model enzyme in solid starch hydrolysis studies. RA is particularly efficient on native maize starch and release glucose only. The hydrolysis rate reaches 75% for a 31% starch solution and is complete at 0.1% starch concentration. The final hydrolysis rate was dependent on both starch concentration and enzyme amount applied. RA is also very efficient in hydrolyzing the crystalline domains in the maize starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in the first stages of hydrolysis. This is in agreement with the observed preferential hydrolysis of amylopectin, the starch constituent that forms the backbone of the crystalline part of the granule. Amylose-lipid complexes present in most cereal starches are degraded in a second stage, yielding amylose fragments that then reassociate into B-type crystalline structures, forming the final resistant fraction.     

Proteomics reveals that redox regulation is disrupted in patients with ethylmalonic encephalopathy

Deficiency of the sulfide metabolizing protein ETHE1 is the cause of ethylmalonic encephalopathy (EE), an inherited and severe metabolic disorder. To study the molecular effects of EE, we performed a proteomics study on mitochondria from cultured patient fibroblast cells. Samples from six patients were analyzed and revealed seven differentially regulated proteins compared with healthy controls. Two proteins involved in pathways of detoxification and oxidative/reductive stress were underrepresented in EE patient samples: mitochondrial superoxide dismutase (SOD2) and aldehyde dehydrogenase X (ALDH1B). Sulfide:quinone oxidoreductase (SQRDL), which takes part in the same sulfide pathway as ETHE1, was also underrepresented in EE patients. The other differentially regulated proteins were apoptosis inducing factor (AIFM1), lactate dehydrogenase (LDHB), chloride intracellular channel (CLIC4) and dimethylarginine dimethylaminohydrolase 1 (DDAH1). These proteins have been reported to be involved in encephalopathy, energy metabolism, ion transport, and nitric oxide regulation, respectively. Interestingly, oxidoreductase activity was overrepresented among the regulated proteins indicating that redox perturbation plays an important role in the molecular mechanism of EE. This observation may explain the wide range of symptoms associated with the disease, and highlights the potency of the novel gaseous mediator sulfide.     

A BAC transgenic Hes1-EGFP reporter reveals novel expression domains in mouse embryos

Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative state of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)^1Hri, to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates some previous observations on Hes1 expression and suggests new, hitherto unrecognised expression domains including expression in the definitive endoderm at early somite stages before gut tube closure and thus preceding organogenesis. This mouse line will be a valuable tool for studies addressing the role of Hes1 in a number of different research areas including organ specification, development and regeneration.     

Schizosaccharomyces pombe encodes a mutated AP endonuclease 1

Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are among the most frequent lesions in DNA. Repair of AP sites is initiated by AP endonucleases and most organisms possess two or more of these enzymes. Saccharomyces cerevisiae has AP endonuclease 1 (Apn1) as the major enzymatic activity with AP endonuclease 2 (Apn2) being an important backup. Schizosaccharomyces pombe also encodes two potential AP endonucleases, and Apn2 has been found to be the main repair activity, while Apn1 has no, or only a limited role in AP site repair. Here we have identified a new 5' exon (exon 1) in the apn1 gene and show that the inactivity of S. pombe Apn1 is due to a nonsense mutation in the fifth codon of this new exon. Reversion of this mutation restored the AP endonuclease activity of S. pombe Apn1. Interestingly, the apn1 nonsense mutation was only found in laboratory strains derived from L972 h(-) and not in unrelated isolates of S. pombe. Since all S. pombe laboratory strains originate from L972 h(-), it appears that all experiments involving S. pombe have been conducted in an apn1(-) mutant strain with a corresponding DNA repair deficiency. These observations have implications both for future research in S. pombe and for the interpretation of previously conducted epistatis analysis.     

A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions

Macroautophagy is a catabolic process that maintains cellular homeostasis and protects cells against various external stresses including starvation. Except for the identification of the Akt-mTORC1 pathway as a major negative regulator, little is known about signaling networks that control macroautophagy under optimal growth conditions. Therefore, we screened a human kinome siRNA library for siRNAs that increase the number of autophagosomes in normally growing MCF-7 human breast carcinoma cells, and identified 10 kinases as regulators of constitutive macroautophagy. Further analysis of these kinases with respect to the autophagic flux, kinase signaling and endolysosomal function identified WNK2 as a positive regulator of autophagosome maturation and nine others as macroautophagy inhibitors. The depletion of MK2, PACSIN1, DAPK2, CDKL3 and SCYL1 functioned upstream of Akt-mTORC1 pathway, whereas CSNK1A1, BUB1, PKLR and NEK4 suppressed autophagosome formation downstream or independent of mTORC1. Importantly, all identified kinases except for BUB1 regulated macroautophagy also in immortalized MCF-10A breast epithelial cells. The kinases identified here shed light to the complex regulation of macroautophagy and open new possibilities for its pharmacological manipulation.