The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education.

Natural killer (NK) cell responsiveness in the mouse is determined in an education process guided by inhibitory Ly49 and NKG2A receptors binding to MHC class I molecules. It has been proposed that inhibitory signalling in human NK cells involves Abl-1 (c-Abl)-mediated phosphorylation of Crk, lowering NK cell function via disruption of a signalling complex including C3G and c-Cbl, suggesting that NK cell education might involve c-Abl. Mice deficient in c-Abl expression specifically in murine NK cells displayed normal inhibitory and activating receptor repertoires. Furthermore, c-Abl-deficient NK cells fluxed Ca2+ normally after triggering of ITAM receptors, killed YAC-1 tumour cells efficiently and showed normal, or even slightly elevated, capacity to produce IFN-γ after activating receptor stimulation. Consistent with these results, c-Abl deficiency in NK cells did not affect NK cell inhibition via the receptors Ly49G2, Ly49A and NKG2A. We conclude that signalling downstream of murine inhibitory receptors does not involve c-Abl and that c-Abl plays no major role in NK cell education in the mouse.

High Dose Rate Brachytherapy for Inoperable Endometrial Cancer: a Case Series and Systematic Review of the Literature.

Endometrial cancer is a common gynaecological cancer, is typically early stage and treated with surgery. For patients where surgery is difficult or dangerous, definitive radiation therapy is the next best option. This study included a single institution case series (step 1) and a systematic review of the literature (step 2). In step 1, all endometrial cancer cases that were treated with definitive image-guided brachytherapy at a single institution from 2008 to 2020 were retrospectively analysed. In step 2, a systematic review of Medline (PubMed) from 1975 to 2020 was carried out using the key words around endometrial cancer and brachytherapy, followed by a narrative synthesis. In total, in step 1, 31 cases were included in this study, stages I-IV, with 96.7% receiving external beam radiation. All patients received three fractions of 7.5 Gy or five fractions of 6 Gy high dose rate brachytherapy, with a median EQD2 of 75.55 (40-84.3). The 2-year Kaplan-Meier (KM) local control was 83.1% and the 2-year KM overall survival was 77.4%. There was no late toxicity ≥grade 3. In step 2, 19 articles were included in the final analysis, with between six and 280 patients. The local control ranged from 70 to 100%, with low toxicity. Definitive radiation therapy with image-guided brachytherapy seems to have good local control with low toxicity for patients who are poor surgical candidates.

A fast, fully automated cell segmentation algorithm for high-throughput and high-content screening.

High-throughput, high-content screening (HT-HCS) of large compound libraries for drug discovery imposes new constraints on image analysis algorithms. Time and robustness are paramount while accuracy is intrinsically statistical. In this article, a fast and fully automated algorithm for cell segmentation is proposed. The algorithm is based on a strong attachment to the data that provide robustness and have been validated on the HT-HCS of large compound libraries and different biological assays. We present the algorithm and its performance, a description of its advantages and limitations, and a discussion of its range of application.

IFN-gamma production and degranulation are differentially regulated in response to stimulation in murine natural killer cells.

Activation of natural killer (NK) cells is induced via receptors like NKG2D, NKR-P1C and NKp46. This activation is balanced by interactions with inhibitory receptors. NK cell activation can lead to cytotoxicity mediated via polarized exocytosis of secretory lysosomes (degranulation) and interferon (IFN)-gamma production. We studied cell surface mobilization of a molecule present in secretory lysosomes, CD107a (LAMP-1), to monitor the relationship between degranulation of NK cells and their production of IFN-gamma at the single cell level. A comparison of responses in naive mouse NK cells and NK cells pre-activated with the type I interferon-inducer tilorone demonstrated a dramatic influence of pre-activation, allowing potent degranulation and IFN-gamma responses to NKG2D mediated stimulation that were not observed with naive NK cells. Degranulation and IFN-gamma production were performed by overlapping NK cell populations with generally higher frequencies of degranulating than IFN-gamma producing NK cells. An NK cell subset analysis based on expression of Mac-1 and CD27 revealed that immature NK cells (Mac-1(lo) CD27(hi)) are preferentially degranulating, Mac-1(hi) CD27(hi) cells perform both effector functions efficiently, while the most mature (Mac-1(hi) CD27(lo)) NK cells display reduced degranulation but with maintained IFN-gamma production.

Critical role of Qa1b in the protection of mature dendritic cells from NK cell-mediated killing.

Molecular interactions in natural killer (NK) cell-mediated killing of dendritic cells (DC) have under recent years come under scrutiny. Upon stimulation with IFN-gamma or lipopolysaccharide, DC become relatively resistant to NK cell-mediated lysis. In the present study, we investigated the role of Qa1(b) on DC and its receptor NKG2A on NK cells in the protection of mature DC from NK cells. We demonstrate that while both NKG2A+ and NKG2A- NK cells can efficiently lyse unstimulated DC, NKG2A+ NK cells but not NKG2A- NK cells are largely impaired in their ability to lyse mature DC. Similarly, mature DC from mice expressing H-2D(b), whose leader peptide sequence binds and stabilizes Qa1(b), were resistant to NK cell-mediated killing, suggesting that stable Qa1(b) expression contributes to the protection of mature DC. This finding was further validated by the demonstration that addition of the Qdm leader peptide could protect TAP1-/- DC from NK cell-mediated lysis both in vitro and in vivo. The present data suggest that stable expression of Qa1 on the surface of mature DC contributes to the protection of DC from NK cell-mediated lysis.

Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions.

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.

Optimization of alternate-strand triple helix formation at the 5"-TpA-3" and 5"-ApT-3" junctions.

Alternate-strand triple helix formation was optimized at the two junction steps, the 5"-TpA-3" and 5"-ApT-3" junctions. Footprint experiments, gel retardation assays and thermal denaturation measures on a sequence appropriately designed with two adjacent alternate-strand polypurine tracts points out that the addition of an adenine residue and the removal of one nucleotide should facilitate the crossing strands at the 5"-TpA-3" junction and at the 5"-ApT-3" junction, respectively. These results provide a 'switch code' for the construction of alternate-strand triple helix forming oligonucleotides which open new possibilities for extending the range of applications of antigene strategy.

Branched oligonucleotide-intercalator conjugate forming a parallel stranded structure inhibits HIV-1 integrase.

Integration of a DNA copy of the HIV-1 genome into chromosomal DNA of infected cells is a key step of viral replication. Integration is carried out by integrase, a viral protein which binds to both ends of viral DNA and catalyses reactions of the 3'-end processing and strand transfer. A 3'-3' branched oligonucleotide functionalised by the intercalator oxazolopyridocarbazole at each 5'-end was found to inhibit integration in vitro. We show that both a specific (G,A) sequence and the OPC intercalating agent contribute to the capability of the branched oligonucleotide to form a parallel stranded structure responsible for the inhibition.

Inhibition by local anaesthetic drugs at low and high stimulation frequencies. A comparison between the isolated phrenic nerve of the rat and the phrenic nerve-diaphragm preparation.

The effects of lidocaine (0.25 mM), prilocaine (0.3 mM), diphenylhydantoin (0.24 mM), tetracaine (0.005 mM) and dibucaine (0.002 mM) on the isolated rat phrenic nerve, neuromuscular transmission and the directly stimulated rat diaphragm, were observed at low (twitch) and high (tetanic) stimulation frequency. The phrenic nerve compound action potential and muscle tension during indirect and direct stimulation were compared. At high frequency stimulation all drugs caused high frequency inhibition of both nerve and muscle. The high frequency inhibition was usually biphasic with an initial decrease in the amplitude of compound action potential and tetanic tension and a subsequent stabilization at a reduced plateau level. The duration of the initial phase increased with the potency of the drugs and was similar for nerve and muscle. A specific high frequency inhibition during indirect stimulation was found with diphenylhydantoin and prilocaine, indicating pre- and post-synaptic inhibition of neuromuscular transmission. At low frequency stimulation, the drugs induced a basal inhibition of the nerve compound action potential. In spite of that, the twitches were not depressed during indirect stimulation of the muscle, illustrating the margin of safety with neuromuscular transmission. These results indicate that the action of the drug was similar at the excitable nerve and muscle membranes when stimulated at high frequency, but different at low frequency stimulation.

Effects of salicylates and paracetamol compared to lidocaine on nerve conduction in vitro.

The effects of acetylsalicylic acid, salicylic acid, sodium salicylate and paracetamol on the compound action potentials of the isolated left phrenic nerve of the rat were observed, and compared to that of lidocaine. All drugs depressed the compound action potential in a reversible, dose-dependent way. Lidocaine (inhibition between 0.05 and 10 mM) was more potent than the other drugs and the depression occurred faster. The inhibitory effects of acetylsalicylic acid and salicylic acid were similar to each other (between 1 and 20 mM). Sodium salicylate was less potent (inhibition between 5 and 70 mM). These concentrations cover the plasma levels reported after therapeutic and toxic doses for the salicylates. Inhibition of nerve conduction may therefore contribute to the adverse nervous effects seen after intoxication with salicylates. In contrast, the observed concentrations for inhibition of the compound action potential caused by paracetamol (between 6.7 and 53 mM) were far greater than the plasma concentrations reported after therapeutic doses or after intoxication with paracetamol. Thus, the inhibition of the compound action potential, caused by paracetamol does not contribute to the therapeutic analgesic effects of paracetamol.

Neurotoxic effects of root filling materials on rat phrenic nerve in vitro.

Inhibitory effects of root filling materials on the conduction of action potentials evoked in rat phrenic nerves were evaluated in vitro. Endomethasone and N2 Normal completely and irreversibly inhibited conductance; ProcoSol caused complete but reversible inhibition; Kloroperka N-O caused total inhibition which was sometimes reversed; and AH26 and Diaket showed partial inhibition which was partially reversible.

Physiological regulation of the secretion of histatins and statherins in human parotid saliva.

The small salivary phosphoproteins, histatins and statherins, have important functions in the oral cavity in terms of antimicrobial actions and regulation of calcium phosphate homeostasis. Neither the effects of various physiological stimuli on their secretion nor the nature of the efferent receptor involved in the stimulus-secretion coupling has been determined previously. These aspects are important for improved understanding of the secretory control of salivary proteins and may have implications regarding the effects of specific medications on salivary constituents and oral health. The effects of graded mechanical (chewing on short and long silicone tubings) and gustatory stimulation (0.5, 1.5, and 5.0% citric acid) on the secretion of histatins and statherins were studied in the presence and absence of adrenolytic agents (n = 10). In this model, secretory rates of both proteins increased with increases in flow rate, with 5.0% citric acid representing a particularly potent stimulus. Histatin and statherin secretory rates were significantly reduced by the beta 1-adrenolytic agent (histatins to 58 to 72% and statherins to 11 to 29% of that in corresponding control experiments), but not by the alpha 1-adrenolytic agent. Since the beta 1-adrenergic receptors played an important role in the stimulus-secretion coupling of these proteins, protective salivary functions in the oral cavity may be compromised during beta 1-adrenolytic treatment.

Effects of formaldehyde on the isolated phrenic nerve and the phrenic nerve-diaphragm preparation of the rat, in vitro.

Formaldehyde, 0.5-4.5 mM, increased the threshold for electrical excitation of the nerve, and led to a partial and reversible inhibition of the compound action potential (cAP). The depression was not enhanced by high frequency stimulation. At 8.9 mM or higher, the depression of the nerve excitability could not be reversed. The inhibition of the nerve developed more slowly than that of the muscle, and the nerve was unaffected after 10 min exposure to 2.2 mM. Formaldehyde, 2.2 mM, caused an immediate depression of the indirectly (through the nerve) and directory (at the muscle) elicited twitch tension. After 10 min the tensions were reduced to respectively, 56% and 49% of control. However, the electromyogram was not changed, indicating that the effect was localized to the excitation-contraction coupling. Tetanic tension (100 Hz in 5 sec) was inhibited more than twitch tension during indirect stimulation, whereas the opposite was found during direct stimulation of the muscle. Thus, during high frequency stimulation, formaldehyde must have an additional effect on the neuromuscular transmission. This effect was localized presynaptically since a fall out of endplate potentials was observed in the formaldehyde-treated diaphragm. In 6.7 mM or higher concentrations the directly or indirectly induced contractions were irreversibly blocked. The resting membrane potential of the muscle cells was unchanged after exposure to formaldehyde. Formaldehyde caused myotonia-like contractions of the diaphragm, occasionally after exposure to low concentrations (2.2 mM), and always after exposure to higher concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential inhibition of A, B and C fibres in the rat vagus nerve by lidocaine, eugenol and formaldehyde.

Evoked compound-action potentials (cAP) in all three fibre types of the isolated nerve were reversibly depressed by exposure to the drugs. After 20 min exposure, lidocaine (0.5 mM) depressed the amplitude of the A and B fibre potentials significantly more than the C fibre potential. With eugenol (0.8 mM), there was no significant difference in depression of A, B and C fibre activity. With formaldehyde (4.5 mM), the latency was longer, and the C fibres were initially significantly more depressed than the A and B fibres. As dull pain from a chronically-inflamed pulp is believed to be mediated through C fibres, these results may explain the difficulties in obtaining complete local analgesia with lidocaine. They indicate that application of low concentrations of eugenol and formaldehyde to the pulp-dentine organ may have an analgesic effect on pain mediated through both A and C fibres.

Effects of eugenol on rat phrenic nerve and phrenic nerve-diaphragm preparations.

The compound-action potential (cAP) of the nerve evoked at 1 Hz was reversibly inhibited and the threshold for nerve stimulation increased between 0.065 and 0.97 mM eugenol. The inhibition showed a sigmoid log10 dose-response relationship. With 2.44 mM or higher concentrations the block became irreversible. The inhibition was enhanced during stimulation at 100 Hz in 5 s with 0.487 mM or higher concentrations. The muscle tetanic tension also showed this high frequency inhibition (HFI). After 40-min exposure to 0.65 mM eugenol, the twitch contractions were unaffected. The indirectly induced tetanic tension was inhibited to 16.6 per cent of control in the initial and to 0.5 per cent in the terminal phase of a 5-s period of 100-Hz stimulation, whereas the directly-induced tetanic tension was inhibited to 38.0 and 13.6 per cent, respectively. Thus, eugenol had affected the neuromuscular transmission during tetanic stimulation in addition to its effect on the directly-stimulated preparation. Intracellular recordings of the end-plate potential (EPP) and miniature end-plate potential (MEPP) indicated both a pre- and a post-synaptic mechanism of action for eugenol. Experiments with eugenol plus d-tubocurarine showed a synergistic effect between these two drugs, suggesting a curare-like effect by eugenol. The resting membrane potential of the muscle was unaffected by eugenol. The following findings suggest that eugenol is a membrane-stabilizing (local anaesthetic) drug at low concentrations: reversible cAP inhibition, increased threshold, high-frequency inhibition, and unresponsiveness of the resting membrane potential of the muscle to the drug.

Evocation of either excitatory or inhibitory reflex responses in human masseter muscle by electrical stimulation of the lip at varying intensities.

Electrical stimuli at 1 Hz with pulse widths of 0.05, 0.1 and 1 ms with intensities from two to six times sensory threshold (2-6 T) were delivered to the lower lip. The reflex responses were monitored by surface electromyography of the ipsilateral masseter muscle. An excitatory response that was not preceded or followed by an inhibition could be evoked in seven out of ten subjects at intensities below 5 T at all pulse widths. A higher stimulus intensities, the excitation disappeared and/or was preceded by as short-latency inhibition (SLI) or a long-latency inhibition (LLI). The electrical threshold for the excitatory response was statistically lower than the SLI and LLI, especially when longer pulse widths wee used. Three subjects demonstrated a primarily excitatory response, whereas four had a more pronounced inhibitory response. It was concluded that separate populations of myelinated fibres may be responsible for the responses: the lowest-threshold fibres may elicit excitatory responses and fibres with higher thresholds may evoke inhibitory responses. Another possible explanation is that central spatial summation could be responsible for the opening of the inhibitory and excitatory central pathways. The excitatory response may be the result of a reflex pathway similar to that evoked by activation of periodontal mechanoreceptors, and could be responsible for the load compensation mechanisms during chewing and/or positioning of food. The inhibitory responses are well known, and are considered to be a protective reflex.

Conditions for excitatory or inhibitory masseteric reflexes elicited by tooth pressure in man.

The reflex responses evoked by slowly rising pressure "push' stimuli to an upper lateral incisor tooth in human masseter muscle were studied. Factors such as the preload (the static force applied to the tooth by the stimulus probe before the start of the push stimulus) and the shape of the stimulus wave affected the outcome of the reflex response. When the stimulating probe did not apply preload to the tooth before the push stimulus took place, the force profile exhibited a large fast component as the probe took up the 'slack' in the periodontium. The fast component in the force profile was found to be responsible for inducing the inhibitory reflex (sole inhibitory reflex). When preload was applied, the size of the fast component in the force profile was reduced and the change in force rate became slower and smoother. This slower stimulus profile induced the sole excitatory reflex significantly more often (58% vs 21%) and the sole inhibitory reflex significantly less often (15% vs 52%) than in the experiments that used no preload. The shape of the stimulus wave that drove the stimulus probe was also of importance. Provided that a 0.5-N preload was applied to the tooth, the smoothest stimulus wave induced the sole excitatory reflex most often. Fitting a rubber attachment to the up of the probe made the push-force profile even smoother and thence more successful in inducing the sole excitatory reflex. Furthermore, the rubber tip reduced the possibility of the probe slipping off the tooth due to small and unavoidable movements of the participant's head. It is concluded that the periodontal mechanoreceptors can induce both excitatory and inhibitory reflexes on the jaw closers. The excitatory reflex becomes dominant when a smooth force is applied with preload. The inhibitory reflex becomes dominant when fast-force changes are applied on the tooth and/or no preload is used.

Simple reaction-time responses to mechanical and electrical stimuli in human masseter muscle.

The latencies of the simple reaction-time responses for opening and closing of the jaws in response to taps and pushes on teeth and lip shocks were measured in human adults. The reaction times were scored from both the integrated electromyographic (EMG) signal in individual trials, and from the cumulative sum (CUSUM) of the averaged EMG response to 50 stimuli. The mean reaction times for both closing and opening were about 80-90 ms for taps and electrical lip shocks, and about 140 ms for push, measured from the CUSUMs. The reaction times in individual trials were difficult to measure from the EMG signal because of the unsteady baseline. In contrast, the reaction times measured from the CUSUMs were clearly defined by the point at which the record began to move sharply above or below the baseline. However, because of the delays inherent in the CUSUM procedure, they were systematically longer than the means of the trial-by-trial measurements.

Nitrate and nitrite concentrations in human saliva: variations with salivary flow-rate.

Salivary nitrate concentration has often been used as a measure of human intake of nitrate. However, our findings indicate that this is not a reliable indicator because the nitrate concentration varies with salivary flow-rate and thus depends on the sampling procedure. Parotid or whole saliva was collected from up to six volunteers under carefully controlled conditions. The effects of stimulating saliva production by chewing on silicon tube (mechanical stimulation) or by sucking citric acid from cotton wool (gustatory stimulation) were investigated. Chewing decreased the average nitrate (plus nitrite) concentration in whole saliva by 59% and the nitrate concentration in parotid saliva (which does not contain nitrite) by 53%, relative to unstimulated saliva. Citric acid stimulation decreased the average parotid salivary nitrate concentration by 88%. Stimulation of salivary secretion increased the total salivary nitrate output and the extent of reduction of nitrate to nitrite for most subjects. The unstimulated parotid salivary nitrate concentration was, on average, 2.8 times the nitrate plus nitrite concentration in unstimulated whole saliva.

Masseter muscle reflex evoked by tapping on osseointegrated Frialit implants.

Comparison was made of the masseter muscle reflexes evoked by tapping on osseointegrated single-tooth aluminum oxide implants, and on natural teeth in nine patients. Tapping on eight out of nine patients evoked an inhibitory masseter muscle reflex, whereas tapping on all of the natural teeth evoked an inhibitory reflex. The threshold for this reflex was clearly elevated in implants compared to natural teeth. The pathway for the impulses responsible for this reflex and the clinical implications of the elevated threshold are discussed.