Supplemental Digital Content is available in the text IgG4-related disease (IgG4-RD) is a novel systemic disease entity characterized by elevated serum IgG4 and tissue infiltration of IgG4-positive plasma cells accompanied by severe fibrosis. Although recent studies demonstrated that innate immune cells including monocytes and macrophages might promote local fibrosis and IgG4 production, the pathological mechanism remains unclear. In this study, we sought to identify the disease-associated genes, especially innate immune molecules.Gene expression was analyzed by DNA microarray in submandibular glands (SMGs) from patients with IgG4-RD (n = 5), chronic sialoadenitis (CS) (n = 3), and controls (n = 3). Differentially expressed genes (DEGs) were validated by real-time polymerase chain reaction (PCR) and immunohistochemical staining in IgG4-RD (n = 18), CS (n = 4), Sjögren syndrome (n = 11), and controls (n = 10).P < 0.05). Gene Ontology (GO) term analysis indicated that the upregulated DEGs in IgG4-RD encoded proteins involved in T/B cell activation and chemotaxis. PCR validated significantly higher expression of macrophage receptor with collagenous structure (MARCO), a pattern-recognition receptor, in IgG4-RD compared with the other groups (P < 0.01). Immunohistochemical analysis confirmed that the expression pattern of MARCO was similar to that of the M2 macrophage marker CD163.Gene expression patterns in the 3 groups were quite different from each other by the pvclust method and principal components analysis. In IgG4-RD, 1028 upregulated genes and 692 downregulated genes were identified as DEGs (MARCO was identified as a disease-associated molecule in IgG4-RD by DNA microarray. Moreover, M2 macrophages might contribute to the initiation of IgG4-RD via MARCO. IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS), so-called Mikuliçz disease, has been considered to be a subtype of Sjögren syndrome (SS) based on the histopathological similarities in glandular tissue. However, in 2005, Yamamoto et al3 reported that Mikuliçz disease demonstrated high serum IgG4 concentration and the infiltration of IgG4-positive plasma cells in glandular tissue, and was distinguishable from SS.IgG4-related disease (IgG4-RD) is a systemic disease presenting in multiple target organs, including pancreas, salivary and lacrimal glands, lung, thyroid, liver, kidney, aorta, prostate, retroperitoneum, and lymph nodes.h2) cytokines such as interleukin (IL)-4 and IL-13.4 We previously reported that specific Th cytokines, including IL-4, IL-10, and IL-21, contributed to IgG4 production and ectopic GC formation in IgG4-DS.6 In addition, recent studies indicated that abnormal innate immune responses enhanced Th2 immune responses and the immunopathogenesis of IgG4-RD via toll-like receptors (TLRs) expressed by macrophages.7 Our recent data indicated that M2 macrophages might promote Th2 inflammatory responses via IL-10 and CC chemokine ligand (CCL) 18 production.8 Although macrophages might play an effective role in IgG4 production and are involved in the initiation of IgG4-RD, the pathogenic mechanism of IgG4-RD by innate immune reaction remains unclear.Regarding the immunological aspects of this disease, it is well known that IgG4 is induced by T helper type 2  because labial salivary glands (LSGs) in IgG4-RD show greater variability in pathology even from the same patient and less characteristic histological findings, such as severe fibrosis and ectopic GC formation, than SMGs.9; submandibulectomy for CS; and neck dissection for OSCC. IgG4-DS was diagnosed according to both the “comprehensive diagnostic criteria for IgG4-related disease” and “diagnostic criteria for IgG4-DS.”10 All patients had never been treated with corticosteroids or any other immunosuppressants before SMG biopsy. All SMG samples from patients with IgG4-RD showed characteristic histopathological findings including marked infiltration of IgG4-positive plasma cells, severe fibrosis, and formation of multiple ectopic GCs. In contrast, SMGs from OSCC patients were histologically normal and lacked clinical evidence of metastasis and radiation therapy .For DNA microarray analysis, SMG samples were obtained from 6 patients with IgG4-RD , 68.2 ± 5.0 years), 3 patients with chronic sialoadenitis (CS) caused by sialolith , and 3 patients with oral squamous cell carcinoma (OSCC) as a control group  who were referred to the Department of Oral and Maxillofacial Surgery, Kyushu University Hospital between 2010 and 2015. The patients underwent the following procedures: open SMG biopsies for IgG4-DS as described by Moriyama et alThis study design was approved by the Ethics Committee of Kyushu University, Japan, and written informed consent was obtained from all of the patients and healthy controls before inclusion in this study .Total RNA was isolated from SMGs using TRIzol Reagent  and then purified using SV Total RNA Isolation and RNeasy system  according to the manufacturer's instructions. RNA samples were quantified using a NanoDrop ND-1000 spectrophotometer  and the quality of the RNA was confirmed with an Experion automated electrophoresis station .According to manufacturers’ instructions, the complementary RNA was amplified and labeled by Low Input Quick Amp Labeling Kit , and hybridized to SurePrint G3 Human Gene Expression Microarrays 8 × 60K v2 (Agilent Technologies) . All hybridized microarray slides were scanned using an Agilent scanner. Relative hybridization intensities and background hybridization values were calculated using Agilent Feature Extraction Software (ver. 9.5.1.1).2-transformed and normalized by the quantile algorithm with “preprocessCore” library package11 on Bioconductor software.12 We selected the probes that called “Present” flag in at least one sample and used them to extract differentially expressed genes (DEGs), and then applied Linear Models for Microarray Analysis (limma) package of Bioconductor software, and obtained DEGs. To identify up- and downregulated genes, we calculated intensity based P values and ratios from the quantile-normalized signal levels of each probe and compared IgG4-RD and CS patients. Then, we established criteria for regulated genes: upregulated genes, ratio ≥4.0-fold and P < 0.05; downregulated genes, ratio ≤0.25 and P < 0.05. The hierarchical clustering by the pvclust method13 and principal components analysis (PCA) were applied to the DEGs from comparison with IgG4-RD and CS. To determine significantly over-represented Gene Ontology (GO) term categories and significant enrichment of pathways (Enrichment Score >1.3), we used GO annotation and the Web tool, the Database for Annotation, Visualization and Integrated Discovery (DAVID) provided by National Institute of Allergy and Infectious Diseases (http://david.abcc.ncifcrf.gov/home.jsp).15 The TreeMap was generated by REVIGO16 to visualize significant (P < 0.01) GO terms that were obtained from the Functional Annotation Chart of Biological Process terms (BP_FAT category) on DAVID.The raw signal intensities of all specimens were log17 and the American-European Consensus Group criteria for SS.18 Each patient showed objective evidence of salivary gland involvement based on the presence of subjective xerostomia and a decreased salivary flow rate, abnormal findings on parotid sialography, and focal lymphocytic infiltrates in the LSGs. There was no documented history of treatment with steroids or any other immunosuppressants, infection with HIV, HTLV-1, hepatitis B virus, or hepatitis C virus, or sarcoidosis in any of the patients. None of the patients had evidence of malignant lymphoma at the time of the study.Quantitative polymerase chain reaction (PCR) and immunohistochemical staining (IHC) were performed to validate the results of DNA microarray analysis. Total RNA was extracted from SMG samples from 18 patients with IgG4-RD, 11 patients with SS, 3 patients with CS, and 10 controls. SS was diagnosed according to both the Research Committee on SS of the Ministry of Health and Welfare of the Japanese Government (1999)Total RNA was isolated from the whole SMG samples by the acidified guanidinium-phenol-chloroform method. Total RNA samples (1 μg) were prepared and used for the synthesis of cDNA and then RNA was incubated for 1 hour at 42°C with 20 U of RNase inhibitor (Promega Corp.), 0.5 μg of oligo-1218 , 0.5 mM of each deoxyribonucleotide triphosphate (dNTP) (Pharmacia), 10 mM of dithiothreitol (DTT), and 100 U of RNA reverse transcriptase .The resulting cDNA was amplified using Light Cycler Fast Start DNA Master mix SYBR Green III  in a Light Cycler real-time PCR instrument . The mRNA levels of CCL18, chitinase 1 (CHIT1), macrophage receptor with collagenous structure (MARCO), and TLR8 were analyzed. The primer sequences used were as follows: CCL18 (187 bp), forward 5′-AGCTCTGCTGCCTCGTCTAT-3′, reverse 5′-CAGGCATTCAGCTTCAGGTC-3′; CHIT1 (102 bp), forward 5′-ATATGCCAGTGGGCAGTTTC-3′, reverse 5′-CCTGTTTCCAGGCTTCTGAG-3′; MARCO (122 bp), forward 5′-CAATGTTCCAAAGCCCAAGAG-3′, reverse 5′-CCTGCAGATTCAGAACTTGGA-3′; TLR8 (124 bp), forward 5′-AAGCACATCCCAAATGAAGC-3′, reverse 5′-GCAACTCGAGACGAGGAAAC-3′; β-actin (260 bp), forward 5′-GCAAAGACCTGTACGCCAAC-3′, reverse 5′-CTAGAAGCATTTGCGGTGGA-3′. The relative mRNA levels were calculated after normalizing to the housekeeping gene β-actin.5 Antibodies used included anti-CD68 , anti-CD163 , and anti-CD123  mouse monoclonal antibodies; anti-CD11c  and anti-CD21  rabbit monoclonal antibodies and a rabbit anti-MARCO polyclonal antibody . Tissue sections were sequentially incubated with primary antibodies for 2.5 hours then with biotinylated anti-mouse IgG or anti-rabbit IgG secondary antibodies , avidin–biotin–horseradish peroxidase complex (Vector Laboratories), and 3,3′-diaminobenzidine (Vector Laboratories). Mayer hematoxylin was used for counterstaining. Photomicrographs were obtained using a light microscope equipped with a digital camera .Serial 4-μm-thick sections of SMGs were cut from the block of formalin-fixed and paraffin-embedded tissue and stained with a conventional avidin–biotin complex technique as previously described.For double immunohistochemical analysis, the formalin-fixed and paraffin-embedded sections of SMGs were immunohistochemically stained using an automated Bond Max stainer (Leica Biosystems). The following primary antibodies were used: anti-CD163  and anti-MARCO . Images were taken using a Keyence BZ-9000 series microscope.2 test, Student t test, Mann–Whitney U test, Kruskal–Wallis tests, and Spearman rank correlations. A P-value of P < 0.05 was considered statistically significant. All statistical analyses were performed using JMP software .The significance of differences between groups was determined using χTable Once we determined that gene expression patterns in patients with IgG4-RD and CS differed from those in controls by clustering analysis, we next compared gene expression and identified genes differentially expressed between IgG4-RD and CS in pairwise comparisons. A total of 1028 genes were identified as upregulated in IgG4-RD compared with CS by the rank products method (see Supplementary Table 1). In contrast, 692 genes were identified as downregulated in IgG4-RD compared with CS (see Supplementary Table 2).Significantly enriched GO terms were found in the 1720 genes extracted as up- and downregulated DEGs in IgG4-RD compared with CS as analyzed by the web tools DAVID. Of the 266 different categories by Gene annotation enrichment analysis and the TreeMap, the following 16 categories were identified as involved in IgG4-RD; immune responses, cell cycle, regulation of leukocyte proliferation, nuclear division, macrophage, cell–cell signaling, cell activation, cell adhesion, DNA replication, ectoderm development, organelle localization, microtubule-based process, di- or tri-valent inorganic cation homeostasis, biological adhesion, cellular di- or tri-valent inorganic cation homeostasis, mitosis .Several genes with GO terms including immune, inflammatory, or macrophage were in the top 120 upregulated DEGs in IgG4-RD compared with CS (Supplementary Table 1). We chose 4 innate immunity-related genes  and validated these genes using real-time PCR. β-actin was an appropriate internal control because the mRNA expression level of β-actin was not significantly different between IgG4-RD and CS in the DNA microarray analysis.8 already demonstrated that CCL18 secreted preferentially by M2 macrophages promotes the development of severe fibrosis in IgG4-DS. In this study, we therefore focused on MARCO as a disease-related molecule.As shown in Figure The SMG samples from patients with IgG4-RD, CS, and SS, and controls were immunohistochemically examined to evaluate the distribution of MARCO. Expression of MARCO could be strongly detected around ectopic GCs only in patients with IgG4-RD, but was rarely seen in the other groups Figure .As MARCO is considered to be expressed on both macrophages and dendritic cells (DCs), we next examined the localization of MARCO and the following markers of macrophages and DCs: CD68 as a marker of both M1 and M2 macrophages; CD163 as a marker of M2 macrophages; CD11c as a marker of myeloid DCs; CD123 as a marker of plasmacytoid DCs; and CD21 as a marker of follicular DCs, in SMG from patients with IgG4-RD.As shown in Figure As mentioned above, IgG4-RD shows predominant infiltration of M2 macrophages over M1 macrophages in the local lesions. Therefore, to clarify whether M2 macrophages expressed MARCO, double staining with CD163 and MARCO was performed. Notably, CD163-positive cells (brown) were colocalized with MARCO-positive cells (red) only in SMGs from patients with IgG4-RD Figure .20 Although IgG4-RD has been considered to be predominantly a Th2 immune response-mediated disease23 recent studies indicated that the initiation of IgG4-RD might be caused by not only acquired immunity, but also innate immunity.25 Watanabe et al7 demonstrated that abnormal innate immune responses induced via TLR signaling in monocytes/macrophages might enhance the immunopathogenesis of IgG4-RD. However, the detailed mechanism of pathogenesis by the innate immune system remains to be completely elucidated. Therefore, we performed gene expression microarray analysis to identify innate immune molecules, especially related to macrophages, involved in the initiation of IgG4-RD. We have previously analyzed the gene expression patterns in LSG samples from patients with IgG4-RD and SS by DNA microarray.26 However, SS is a unique inflammatory disease similar to IgG4-RD, while CS is a nonspecific inflammatory disease, suggesting that CS is suitable as positive controls for IgG4-RD.IgG4-RD is a new emerging disease entity that has received much attention recently. Several studies have already reported the clinical features and symptoms of this disease.Microarray of SMGs demonstrated significant differences in gene expression patterns among the patients with IgG4-RD, CS, and controls. According to the results of Functional Annotation Clustering on DAVID, we found that the terms related with immune response, inflammatory response, and macrophage were enriched in IgG4-RD compared with CS. Thus, IgG4-RD might be caused by some specific inflammatory milieu and these results are consistent with past reports with regard to the pathogenesis of IgG4-RD.h2 cells.27 We have already reported that CCL18 was associated with the development of severe fibrosis in IgG4-RD.8 In addition, previous data, indicating that tissue-infiltrating and circulating IgG4-positive cells in IgG4-RD are polyclonal,28 suggest that there are many antigen responses in local lesions. Thus, we focused on pattern recognition receptor MARCO as a new candidate molecule related to innate immunity in this study.In this study, overexpression of CCL18 and MARCO in SMGs from patients with IgG4-RD compared with the patients with CS, SS, and controls was validated by real-time PCR. CCL18 was identified previously as a chemokine with fibrotic activity and selective chemotactic activity on peripheral blood T lymphocytes, especially T29 and is considered to play an important role in the innate immune response by mediating ligand binding and phagocytosis. This receptor can recognize various ligands including bacterial lipopolysaccharide (LPS), acetylated low density lipoprotein (LDL), apoptotic cells, lung pathogens, Streptococcus species, and ozone-generated oxidized lipids, environmental particles, and nanomaterials such as carbon nanotube and silica.30 The expression of MARCO on alveolar macrophages was reported to be strongly increased by exposure to carbon nanotubes, which were similar in structure to asbestos.31 Macrophages are well known as key regulators of the immune system due to recognizing pathogen antigens, phagocytosing them and presenting them to lymphocytes. There are at least 2 major subsets of macrophages; classically activated (M1) macrophages stimulated by Th1-type responses and alternatively activated (M2) macrophages stimulated by Th2-type responses.32.M2 macrophages scavenge debris and contribute to angiogenesis, suppression of adaptive immunity, wound healing and fibrosis by producing IL-10 and CCL18.33 Murthy et al34 found that alveolar macrophages from patients with asbestos-induced pulmonary fibrosis showed high expression levels of MARCO. These observations suggested that MARCO promotes the polarization to a profibrotic M2 phenotype and causes an excessive fibrotic response to lung injury.MARCO was identified as a pattern-recognition receptor belonging to the scavenger receptors expressed by macrophages,8 In addition, IgG4-RD patients showed strong infiltration of DCs. Several studies reported that increased DCs might play a pathological role in IgG4-RD.36 However, additional research is required to further elucidate the involvement of these cells in the pathogenesis of IgG4-RD.Immunohistochemical analysis in our present study found that MARCO was strongly expressed around ectopic GC only in IgG4-RD patients and was colocalized with CD163-positive cells (M2 macrophages). Our previous data indicated that IgG4-RD patients showed a predominant infiltration of M2 macrophages in multiple lesions including those of the pancreas, pleura, prostate glands, lacrimal glands, and SGs.Considering the current results, M2 macrophages may contribute to the initiation or maintenance of IgG4-RD via MARCO. We thus made the following hypothesis regarding the pathogenic process in IgG4-RD; M2 macrophages recognize certain exogenous or endogenous molecules through binding to MARCO which promote the production of factors, including IL-10 and CCL18, which precipitate an exaggerated fibrosis and the pathology noted in IgG4-RD .In conclusion, we have confirmed that MARCO is overexpressed in M2 macrophages clustered around ectopic GCs. However, we need to validate the mRNA expression levels of MARCO and the other DEGs related to innate immunity in many more patients, and perform functional assays to elucidate the relationship between innate immunity and IgG4-RD. A more thorough understanding of the role of innate immune cells in IgG4-RD, especially M2 macrophages, could lead to the establishment of a mouse model of IgG4-RD and the development of novel pharmacological strategies aimed at disrupting the innate immune network and inhibiting the initiation or progression of IgG4-RD.


V2.1 (P/Q-type) Ca2+ channels. Knockin (KI) mice carrying the FHM1 R192Q missense mutation show enhanced cortical excitatory synaptic transmission at pyramidal cell synapses but unaltered cortical inhibitory neurotransmission at fast-spiking interneuron synapses. Enhanced cortical glutamate release was shown to cause the facilitation of cortical spreading depression (CSD) in R192Q KI mice. It, however, remains unknown how other FHM1 mutations affect cortical synaptic transmission. Here, we studied neurotransmission in cortical neurons in microculture from KI mice carrying the S218L mutation, which causes a severe FHM syndrome in humans and an allele-dosage dependent facilitation of experimental CSD in KI mice, which is larger than that caused by the R192Q mutation. We show gain-of-function of excitatory neurotransmission, due to increased action-potential evoked Ca2+ influx and increased probability of glutamate release at pyramidal cell synapses, but unaltered inhibitory neurotransmission at multipolar interneuron synapses in S218L KI mice. In contrast with the larger gain-of-function of neuronal CaV2.1 current in homozygous than heterozygous S218L KI mice, the gain-of-function of evoked glutamate release, the paired-pulse ratio and the Ca2+ dependence of the excitatory postsynaptic current were similar in homozygous and heterozygous S218L KI mice, suggesting compensatory changes in the homozygous mice. Furthermore, we reveal a unique feature of S218L KI cortical synapses which is the presence of a fraction of mutant CaV2.1 channels being open at resting potential. Our data suggest that, while the gain-of-function of evoked glutamate release may explain the facilitation of CSD in heterozygous S218L KI mice, the further facilitation of CSD in homozygous S218L KI mice is due to other CaV2.1-dependent mechanisms, that likely include Ca2+ influx at voltages sub-threshold for action potential generation.Familial hemiplegic migraine type 1 (FHM1) is caused by gain-of-function mutations in Ca V2.1 (P/Q-type) Ca2+ channels play a prominent role in initiating action potential (AP)-evoked neurotransmitter release at brain excitatory and inhibitory synapses  mice  from KI mice that carry either the FHM1 R192Q or the S218L mutation  compared with homozygous R192Q (RQ/RQ) KI mice  KI mice were similar to those in RQ/RQ KI mice  whether the gain-of-function of evoked glutamate release in SL/WT and SL/SL KI mice correlates with the similar gain-of-function of the P/Q-type Ca2+ current and facilitation of CSD in SL/WT and RQ/RQ KI mice and with the larger gain-of-function of the P/Q-type Ca2+ current and facilitation of CSD in SL/SL than SL/WT and RQ/RQ KI mice; and (iii) whether, as a consequence of the particularly low threshold of activation of mutant S218L channels, a fraction of these channels are open at resting membrane potential in cortical glutamatergic synaptic terminals.The effect of other FHM1 mutations, including the more severe S218L mutation, on cortical synaptic transmission remains unknown. Here, we studied excitatory and inhibitory neurotransmission in microcultures of cortical neurons from neonatal S218L KI and WT mice to investigate: (i) whether the S218L mutation, like the R192Q mutation, enhances excitatory neurotransmission by increasing AP-evoked CaCacna1a gene were generated as previously described  with properties very similar to those of synapses between neurons; these autaptic connections are by definition monosynaptic, offer an unusually homogeneous population of synapses producing large synaptic responses and solution exchanges can be fast and complete .A-mediated IPSCs recorded at −90 mV are inward currents , they were easily distinguished from glutamate receptor-mediated EPSCs on the basis of their slower time course and their complete inhibition by 20 µM bicuculline (Ascent Scientific-Abcam). The currents recorded in the presence of bicuculline were subtracted to all records to obtain the evoked IPSCs (displayed in the figures after blanking 1–2 ms around each stimulus artifact for clarity). After PSC stabilization , 5–10 sweeps were averaged to obtain the PSC amplitude.Microislands containing several glial cells and a single neuron were selected for recording of evoked postsynaptic currents (PSCs) in voltage-clamp mode  after 8–14 days (DIVs) in culture. APs in the unclamped processes were induced by a 2 ms voltage pulse to +20 mV every 10 s from a holding potential of −80 mV. The evoked PSCs were measured at −80 mV and −90 mV for excitatory and inhibitory PSC, respectively. Single neurons with a pyramidal cell morphology characterized by a typical prominent process emanating from a triangular/pyramidal soma were selected for recording glutamate receptor-mediated excitatory postsynaptic currents (EPSCs), as in Tottene et al. . The cur2, 30 HEPES, 3 EGTA, 4 ATP, 0.5 GTP and 1 cAMP (pH 7.4 with KOH). The extracellular solution contained (in mM): 145 NaCl, 3 KCl, 10 HEPES, 10 glucose, 1 MgCl2, 2 CaCl2, 0.05 D-AP5 (pH 7.4 with NaOH). In the experiments testing the effect of the peptide toxin ω-AgaIVA (Aga) (Peptide Institute Inc.), cytochrome C (0.1 mg/mL) was added to the solution.The pipette solution contained (in mM): 110 K-methane-sulfonate, 5 MgClA liquid junction potential (LJP) of −8 mV should be added to all voltages to obtain the correct membrane potentials Neher, . Patch-cThe relative number of neurons recorded from WT and KI mice at different DIVs was closely matched.3, 1.25 NaH2PO4, 0.5 MgCl2, 1 CaCl2, 25 glucose saturated with 95% O2 5% CO2. In order to record mEPSCs, after the break-in and the recording of the firing pattern, slices were superfused with the same ACSF solution containing (in µM): TTX 0.2, D-AP5 50, bicuculline 20 and cytochrome C (0.1 mg/mL). mEPSCs were acquired at −69 mV for at least 3 min  before and after application of 400 nM Aga and analyzed using Clampfit 10.0 (Axon Instruments), as in Tottene et al. .Data are given as mean ± SEM; stars indicate a statistically significant difference from control assessed by the unpaired or paired Student’s 2+ influx and increased glutamate release at cortical pyramidal cell synapses, we studied excitatory neurotransmission in cortical neurons from neonatal SL/WT KI and WT mice grown in microculture. EPSCs were evoked in single cortical pyramidal cells by brief depolarizing voltage steps eliciting an AP in the unclamped axonal processes .To assess whether the FHM1 S218L mutation leads to increased AP-evoked Cas Figure  have pre2+ influx through P/Q-type Ca2+ channels located at the active zones and, as a consequence, to increased probability of vesicle release, one predicts a larger AP-evoked EPSC amplitude and a larger contribution of P/Q-type Ca2+ channels to neurotransmission in SL/WT KI relative to WT mice, as previously found in RQ/RQ KI mice  in KI compared with WT neurons  was similar to the difference in size of the corresponding total EPSC amplitude  in SL/WT vs. 1.65 ± 0.10 mM (n = 7) in WT; p = 1.0 × 10−5)  Figure , as prev) Figure .Moreover, the finding that the paired-pulse ratio  was lower in SL/WT KI than WT mice Figure  is consi2+ channels to neurotransmission as well as the Ca2+ dependence of the EPSC and the PPR are quantitatively similar in cortical pyramidal cell autapses of heterozygous SL/WT KI and homozygous RQ/RQ KI mice. In fact, according to Tottene et al. . The similar increase in AP-evoked Ca2+ influx and probability of release in SL/WT and RQ/RQ KI mice correlates with the relatively small difference in gain-of-function of the CaV2.1 current at low voltages in neurons from these mice  and EC50 = 0.90 ± 0.07 mM for SL/WT KI (n = 11); p = 0.26) and, in a larger set of experiments, the ratio of the EPSCs evoked at 1 mM and 2 mM [Ca2+]out was also similar in SL/SL and SL/WT KI mice (0.55 ± 0.04 in SL/SL (n = 21) and 0.61 ± 0.04 in SL/WT (n = 17); p = 0.23). If, as expected from the larger gain-of-function of the neuronal CaV2.1 current in SL/SL relative to SL/WT KI mice , then the Ca2+ dependence of the EPSC at SL/SL autapses should be shifted to lower [Ca2+]out relative to that at SL/WT KI autapses. The finding of a similar Ca2+ dependence of the EPSC in SL/SL and SL/WT KI mice is consistent with the existence of compensatory changes like those mentioned already and/or with a compensatory decrease in the number of functional CaV2.1 channels at the active zones in SL/SL KI mice (see Section Discussion). Whatever the exact underlying compensatory mechanism, the similar gain-of-function of excitatory neurotransmission at cortical pyramidal cell synapses of SL/SL and SL/WT (and RQ/RQ) KI mice contrasts with the larger facilitation of CSD in SL/SL compared to SL/WT (and RQ/RQ) KI mice  in both SL/SL (30 ± 4%) and SL/WT (21 ± 4%) cortical slices , lead to hyperactivity of cortical circuits, mainly due to excessive recurrent excitation, that may create the conditions for the initiation of spontaneous CSDs  the dependence of the EPSC on [Ca2+]out was shifted to lower values; and (iii) the PPR was lower in S218L KI than in WT mice. The gain-of-function of both the evoked EPSC and the contribution of P/Q-type Ca2+ channels to neurotransmission as well as the Ca2+ dependence of the EPSC and the PPR at cortical pyramidal cell autapses of heterozygous SL/WT KI mice were quantitatively similar to those reported for homozygous RQ/RQ KI mice  KI mice to compensate for the larger Ca2+ influx through open mutant CaV2.1 channels. Evidence for the latter compensatory mechanism at Calyx of Held terminals of SL/SL KI mice is provided by the finding that, while the gain-of function of the Ca2+ current at low voltages is larger in Calyx terminals from SL/SL than RQ/RQ KI mice, the Ca2+ current at high voltages is reduced in SL/SL but unchanged in RQ/RQ KI compared with WT terminals  and cerebellar granule cell synapses  observed in S218L (both larger in SL/SL than SL/WT) but not R192Q KI mice  KI mice contrasts with the larger facilitation of experimental CSD in SL/SL compared with SL/WT (and RQ/RQ) KI mice . As a trend, the fractional reduction was larger in SL/SL than SL/WT KI mice, but the difference did not reach statistical significance, possibly because even in SL/SL pyramidal cells only 30% of mEPSCs were Aga-sensitive. The compensatory changes that prevent a larger gain-of-function of evoked glutamate release in SL/SL compared with SL/WT KI mice may also reduce the difference in the fraction of Aga-sensitive mEPSCs between the two genotypes, and, in the case of a decreased affinity of presynaptic Ca2+ sensor(s), may account for the similar mEPSCs frequency in SL/SL and SL/WT pyramidal cells.A feature of cortical synaptic transmission in S218L KI mice not present in R192Q KI mice is the presence of a fraction of mutant CaV2.1 channels that are open at rest in cortical excitatory synaptic terminals is much smaller than that at Calyx terminals  that has been proposed to underlie the gain-of-function of AP-evoked glutamate release in Calyx  are key elements in the positive feedback cycle that ignites CSD  KI mice that may prolong the transient hypoxia and slow the recovery of cerebral flow after CSD (Pietrobon and Moskowitz, 2+ channels, besides the particularly low threshold of activation, is the incomplete inactivation during prolonged depolarizations (Tottene et al., in during CSD in cortical neurons may also contribute to a slower recovery from CSD in S218L KI mice. A slower recovery from CSD could provide a plausible explanation for the unique susceptibility to recurrent CSDs (van den Maagdenberg et al., One can envisage several mechanisms by which CaDania Vecchia performed the experiments, analysed the data, discussed findings and interpretation of the data and contributed to drafting the manuscript.Angelita Tottene contributed to the acquisition, analysis and discussion of the data and revised critically the manuscript.Arn M.J.M. van den Maagdenberg contributed to the discussion and interpretation of the data, provided reagents and analytic tools, and revised critically the manuscript.Daniela Pietrobon conceived and designed the study, discussed findings and interpretation of the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phonon optics concerns operations with high-frequency acoustic waves in solid media in a similar way to how traditional optics operates with the light beams (i.e. photons). Phonon optics experiments with coherent terahertz and sub-terahertz phonons promise a revolution in various technical applications related to high-frequency acoustics, imaging, and heat transport. Previously, phonon optics used passive methods for manipulations with propagating phonon beams that did not enable their external control. Here we fabricate a phononic chip, which includes a generator of coherent monochromatic phonons with frequency 378 GHz, a sensitive coherent phonon detector, and an active layer: a doped semiconductor superlattice, with electrical contacts, inserted into the phonon propagation path. In the experiments, we demonstrate the modulation of the coherent phonon flux by an external electrical bias applied to the active layer. Phonon optics using external control broadens the spectrum of prospective applications of phononics on the nanometer scale. SLs are widely used for the generation and detection of coherent phonons in phonon optics experiments; they may be made with semiconductorsThe paper is organized as follows: after describing the phononic chip used in the experiment, we shall present the results of phonon generation and detection with an unbiased device, which is equivalent to a passive phononic chip. Further, we shall turn to the main experimental result of the work where we study the effect of electrical bias in the doped SL on the amplitude of the transmitted coherent phonon signal. Finally, we present analysis of the results, discussion and conclusions. The experiment and analysis details may be found in the Methods section.f0 = 378 GHz are excited by optical pulses from a femtosecond pulsed laser; (ii) an active n-doped GaAs/AlAs SL with heavily n-doped contacts for applying electrical bias; and (iii) a p-i-n detector of coherent phonons on the opposite side of the GaAs substrate to the SLsp-i-n on both sides on the substrate were fabricated in the form of mesas (p-i-n detector. The low temperature current-voltage (I–V) characteristic of the active SL device is shown in The design of the phononic chip and the scheme of the experiment used in the present work is shown in of mesas  with theV is applied, then through the GaAs substrate with thickness of d = 164 μm, and finally are detected using optical probing with temporal resolution ~1 ps in the p-i-n detector. The detected signal VP (t) is monitored as function of the delay t between pump and probe pulses. The VP (t) provides direct information about the temporal evolution of dynamical strain in the p-i-n which accompanies the coherent acoustic wavepacket. The techniques for generation and detection of coherent phonons are well developed so won't be discussed in detail here, details of the current experiment may be found in the Methods section and earlier papers describing generation in a SLp-i-n photodiodeT0 = 10 K to minimise attenuation of the 380 GHz phonons as they propagate through the substrate. The low temperature is also a requirement for the operation of the active SL, and ensures that the thermal broadening of the electronic energy states is no more than the energy of the 378 GHz phonons. In our experiment the wavelength of pump and probe pulses was chosen in the range 690–700 nm to have sufficient absorption by electron-hole (exciton) optical transition in 4 nm GaAs layers and full transparency in 11 nm AlAs barriers which form the transducer SL. Then theoretical estimations predict the generation of quasi-monochromatic acoustic phonons in the transducer SL with the frequency 380 GHz and the duration of the wavepacket ~4 nsIn experiments with the phononic chip the generated phonons pass through the active SL, where the bias P0(t) detected in the p-i-n for the case when the bias is not applied to the active SL (V = 0) is shown in P0(t) is spread over the time interval ~1 ns and includes several high-amplitude peaks and harmonic oscillations with a frequency of f0 = 378 GHz  obtained for the full temporal interval is shown in f0 = 378 GHz and f1 = 519 GHz with the widths 2 and 10 GHz respectively indicated in P0(t) and its spectrum are in agreement with the earlier results in phonon optics experiments with passive SL devices21f0 = 378 GHz that is attributed to coherent phonon generation in the transducer SL as a result of pump optical excitation.With no bias applied to the active SL, the experimental device can be considered to be a passive phononic chip. The temporal evolution of the strain f1 = 519 GHz are generated in the same way as in the top SL. High-amplitude peaks, which form the low frequency part (f < 250 GHz) of the phonon spectrum are due to optically induced stress in various semiconductor layers. Particularly, the peaks in P0(t) with the highest amplitudes correspond to the strain pulses generated in the heavily doped n-contact near the interface with the transducer SL. The arrival times tC and tR of the direct and reflected strain pulses respectively are shown by dotted vertical lines in A small fraction of the optical pump beam is transmitted through the contact GaAs layers and reaches the second SL where phonons with f0 = 378 GHz. The pump light penetrates into the top SL and generates coherent phonons throughout its full thickness, and so the first phonons to reach the detector will be emitted from near the bottom of the SL. As the SL is a total of 750 nm in thickness, these phonons arrive 130 ps earlier than those from the top surface. Therefore, the oscillations with frequency f0 in P0(t) start at ≈-130 ps. These oscillations last for a time longer than 1 ns and possess the highest amplitude in the time interval t = 100 – 200 ps marked by dashed rectangle. This interval will be the subject for further studies of the effect of electrical bias in the active SL on the phonon signal at f0 = 378 GHz.Further we shall concentrate on the monochromatic part of the phonon spectrum at V, to the doped SL on the coherent phonon signal VP(t) we modulate bias voltage V and detect the difference ΔP(t) = VP(t)-P0(t) using a digital lock-in amplifier. The frequency of the bias modulation was set at 15 kHz, which corresponds to the period much shorter than the thermal time constant of the sample, thus avoiding a significant lattice temperature modulation in the chip. This was confirmed by measuring the arrival time of the acoustic wavepacket with bias on and off as is further explained in the discussion section. We present the results in the time interval marked by dashed frame in P(t) for V = 500 mV and P0(t) are shown in P(t) and P0(t), possess temporal evolutions which show oscillatory behaviour. The maximum relative changes ΔP(t)/ P0(t) ~0.14% in the spectral amplitudes at f0 are high enough to detect the signal in the phononic chip using the p-i-n photodiode with sufficient signal to noise level. It is important that the temporal shape of ΔP(t) is not exactly the same as the temporal shape of P0(t), which means that the origin of ΔP(t) is not simply due to a modulation of the p-i-n detector sensitivity by any stray electric field from the biased SL.To study the effect of applying an electrical bias, P0(t) and ΔP(t) are shown in f0 = 378 GHz. The similarity between these two spectra means that the applied bias has an effect on both parts of the spectra: broadband low-frequency part (f ≤ 200 GHz) and monochromatic coherent phonons with f0 = 378 GHz. As mentioned earlier, the major contribution to the broadband low frequency part of the coherent phonon spectrum comes from the strain pulses generated in the n-GaAs contact where bias is applied. It is reasonable to assume that the application of bias changes the widths of the depletion regions in the contacts, which affects the optical absorption length and correspondingly the spectrum of the generated coherent phonons in the acoustic wavepacket of the strain pulse. This effect, while being interesting by itself, however stays beyond the present work, and further we shall concentrate on the contribution from monochromatic phonons with f0 = 378 GHz to ΔP(t). The only source of these monochromatic phonons in the studied phononic chip is the transducer SL excited by the optical pump pulses.The FFT spectra of P0(t) and ΔP(t) transmitted phonon spectra components with cut off frequency at 250 GHz, and show corresponding traces and spectra in f0 = 378 GHz look more distinct and it is possible to compare the phases of the monochromatic contributions in P0(t) and ΔP(t) (see inset in πf0t + Ψ) it is possible to obtain the phase difference ΔΨ = Ψ0 − ΨΔ where Ψ0 and ΨΔ are the phases of the f0 harmonic component P0(t) and ΔP(t) respectively. Performing the fitting procedure in seven 20 ps temporal intervals we get a value of ΔΨ = (0.76 ± 0.03)π which will be discussed in more detail in the next section.For clarity of the presentation we apply high-pass digital filtering to f0 components in the signals P0(t) and ΔP(t). Ignoring any nonlinear effects it is easy to show that the amplitude ratio α = D/A0 (A0 and D are the amplitudes of the P0(t) and ΔP(t) respectively) depends on the bias induced changes of ΔA = V-AA0 (VA is the amplitude of VP(t)) and on the difference Δt between the arrival times of coherent phonons to the p-i-n detector with bias on and off. From the experiment we estimate α ~ 0.14% for V = ± 500 mV and, using the analysis described in the methods section, we deduce that |ΔA/A0| ~ 0.1% and Δt ~ 0.4 fs. These results show the extremely high sensitivity of the modulation technique and p-i-n phonon detection used in this experiment.We start the discussion by considering the relative amplitude and phase of t is most likely to be due to the bias induced modulation of the sound velocity as a result of lattice temperature modulation. Indeed, application of bias to the active SL is accompanied by Joule heat and respectively by an increase of the lattice temperature in the propagation path for phonons from the transducer SL to the p-i-n detector. From the difference in the arrival time of the phonon induced signal at V = 0 and 0.5 V applied bias, we estimate the average sample temperature T1 based on the dependence of the sound velocity c on T in GaAs, i.e. c11 and ρ are the elastic constant and density of GaAs respectively. Using the published temperature dependencies of c11ρT0 = 10 K to the value T1 ≈ 23 K by applying the bias.The non-zero value of ΔT, which, for the case when the modulation period is much less than the thermal time constant of the sample, is (≪ T1). Assuming the measured value of the delay Δt = 0.4 fs is purely due to the temperature modulation, then using the value T ~ 10−3 K. Such a small value of ΔT cannot explain the measured value |ΔA/A0| if the transmission change was due to anharmonic phonon-phonon scattering. Indeed, estimates based on Herring mechanism of anharmonic decayf0 phonon amplitude of not more than 4 × 10−5 which is two orders of magnitude smaller than the experimentally measured |ΔA/A0| ~ 1 × 10−3. Thus we exclude the modulation of the lattice temperature as the reason for the bias induced modulation of the phonon flux amplitude.Modulation of bias with a frequency of 15 kHz leads to modulation of the sample temperature with the amplitude Δf0 = 378 GHz is based on the model of electron-phonon interactions in the active SL. The band diagram of the SL with applied bias, where electrons are well separated in energy and space, is called a Stark ladderFig. 440Our explanation of the bias-induced modulation of the coherent phonon flux with α on V, shown on α on V is very different from the I–V characteristic of the active SL , which is well away from the phononic stop bands of the active SL. The phonon detector based on the p-i-n photodiode was grown by MBE on the opposite side of the substrate. The phonon detector consists of 3 period SL with 1.2 nm of GaAs and 1 nm of Al0.33Ga0.77 As. It is grown in the middle of a 200 nm-thick undoped (i-intrinsic) Al0.33Ga0.77 As layer and has maximum sensitivity to phonons when probed with the optical pulses with photon energy ~1.76 eV, close to the band gap of GaAs layers in the SL. The i-region is embedded between 200 nm thick contacts from n-doped (1018 cm−3 Si) Al0.33Ga0.77 As and p-doped (1018 cm−3 C) Al0.33Ga0.77 As layers. This particular structure was chosen so that its optical resonance matched the optical resonance of the phonon transducer SL, and it was found to be at least as sensitive as the single GaAs quantum well based structureThe semiconductor layers in the chip shown in 2 per pulse. The probe beam was focused on the p-i-n detector to the spot with diameter ~20 μm. The amplified photocurrent of the biased p-i-n was detected by the lock-in amplifier referenced to 15 kHz from the acousto-optical modulator in the path of the pump beam (in experiments with V = 0) or from the pulses controlling the bias modulation in the active SL.Optical pump and probe beams came from a tuneable mode locked Ti:sapphire ~100 fs pulsed laser with a repetition frequency of 82 MHz operating at a wavelength of 700 nm. After passing through the motorized optical delay line the pump beam was focused on the transducer SL to the 50 μm diameter spot and the maximum energy density at the surface of the phononic chip was ~50 μJ/cmP0(t) shows a number of sharp peaks. Three of them are explained in the caption of 0(t)P also possesses other peaks with smaller amplitudes which may be explained by the arrival of the strain pulses generated at various interfaces due to stress induced by optical pump excitation. The total number of peaks also includes the echoes in the p-i-n diode due to the phonon reflection at the open surface from the side of phonon detectorp-i-n photodiode and shown in The signal VP(t) applying dc bias V (without bias modulation) that ΔA/A and 2πf0Δt are both much less than unity. We may get the following equation for the ratio α:t = 0 , then α = |ΔA/A0| and the phase shift ΔΨ = 0 or ΔΨ = π when the applied bias induces the increase or the decrease of the amplitude of harmonic oscillations respectively. In the other extreme case, when ΔA = 0, α = |2πf0Δt| and ΔΨ = ± π/2. In more the general case, Δt≠0 and ΔA≠0, the phase difference is equal to:H(x) is the Heaviside function. A < 0 and this means a decrease of the oscillation amplitude when bias is applied to the active SL. Using the experimentally measured value of ΔΨ and t. Following this 0).We checked experimentally measuring A.J.K. and A.V.A. developed the idea for the experiment, C.L.P. and A.V.A. performed the experiment, C.L.P., A.V.A. and A.J.K. analysed the experimental data and wrote the manuscript, R.P.C. fabricated the samples.


We aimed to report a simple estimation method to enable quantification of pediatric antibiotic exposure in large aggregated datasets. Secondly, we aimed to quantify and benchmark Hungarian pediatric antibiotic use.First we intended to examine whether a correlation existed between dosage form data and the patient’s age. Therefore, issued prescriptions were analyzed in pharmacies. As a correlation was found between the share of liquid oral antibacterial products and the rate of pediatric antibiotic prescriptions , we extrapolated this finding to a large aggregated dataset and estimated that 34.6% of prescriptions were issued for pediatric cases . Taking into account the demography of the population, children were exposed to antibiotics three times more often than adults with a corresponding annual prescription rate of 2.6.We demonstrated that simple drug-related data can be linked to a patient-related measure as we found strong associations between dosage form data and patients’ age. Based on this association, massive pediatric antibiotic exposure was found. Due to the general availability of dosage form data and the ease of the estimation method, the reported concept can be used to quantify pediatric antibiotic use in large aggregated datasets or when age stratification is absent. Antibiotics are amongst the most commonly prescribed medicines to children –3. DespiAs liquid dosage forms are available for many antibiotics, we hypothesized that their consumption can be linked to pediatric antibiotic use and hence drug-related data can be translated to a patient-related measure.The goal of the present work was to report a simple estimation method to enable the quantification of pediatric antibiotic exposure. Secondly, we aimed to quantify and benchmark Hungarian pediatric antibiotic use.During the 120 study-days, around 50,000 prescriptions were dispensed, out of which 2,846 referred to antibacterials. In more than 90% of the cases, one package of antibacterial product was ordered per prescription, with an average of 4.3 DDDs in children and 8.1 DDDs in adults. In total, 1,009 prescriptions (36.1%) were prescribed for children , out of which 11.5% were liquid oral formulations. Applying the result of the linear regression to the aggregated regional dosage form data, it was estimated that 34.6%  of the antibiotic prescriptions were ordered for children, while the rest (65.4%) were prescribed for adults.Considering the population of the children  within the region and their 34.6% share from all antibiotic prescriptions, children were prescribed antibiotics three times more often than adults ((34.6/14.9)/(65.4/85.1)=3.02). Bearing in mind the weighted average DDD content (4.3 vs. 8.1 DDDs) and the proportional rate (34.6% vs. 65.4%) of pediatric and adult prescriptions, the calculated number of pediatric prescriptions were 262,165, which corresponds to an annual pediatric prescription rate of 2.6 . Pediatric antibiotic exposure expressed in DDD/1,000 children inhabitants/year was 31.1 .In the present report, a strong association between liquid oral antibacterial use and the rate of pediatric antibiotic prescriptions is shown. The availability of comprehensive pediatric antibiotic use data are limited . On the Despite the relatively easy computability of the dosage form data, no publication exists on the use of different oral antibiotic formulations. Bronzwaer was the first who expressed the need for age-stratified antibiotic use data and presumed that the analysis of the liquid formulation might help in assessing pediatric use . Liquid In the literature, different measures were used to express antibiotic exposure in children , 10. A pIn summary, compared to the limited pediatric antibiotic use data reported, a relatively frequent exposure of Hungarian children to antibiotics is suggested.A major limitation of this study is the direct application of the revealed correlation coefficient to the foreign dataset. Similar associations between the use of liquid oral dosage forms and age certainly exist, but as the usage of liquid formulations and populations’ age distribution may differ, each country should set up their own linear regression model by the analysis of suitable patient-level sample data. Another handicap worth mentioning is that we calculated the annual pediatric prescription rate by duplicating the half-year data. As the study period covered the winter months, including the influenza epidemics, but excluded the start of the semester in school/kindergarden  with peaks in the incidence of respiratory tract infections, the reported annual pediatric antibiotic use value is a valid estimation. Furthermore, we have only analyzed data of one Hungarian region, not the entire country. Considering that this region covers almost 20% of the area and more than 13% of the population of Hungary, the level of antibiotic use ranks in the middle of Hungarian regions (data not shown), and it is not likely that proportional use of liquid formulations differs largely in other regions, therefore our results reflect the Hungarian situation well.In this study, in the different sample pharmacies, the rate of pediatric prescription varied highly, and the share of liquid formulations ranged substantially. Diversity may be due to the various catchment populations of pharmacies: the population’s age distribution may differ depending on where the pharmacies are located, as for example pharmacies next to a pediatricians’ office dispense more pediatric therapies. Both diversity and the recorded similar share of liquid oral formulations  reflect the representativeness of the sample and validity of our estimation.First and foremost, we intended to examine the correlation between dosage form data and the patient’s age. As both of these data are available on issued prescriptions only, we conducted a study in 20 sample pharmacies. As a correlation was found between these two variables (see details below), results were projected to the regional dataset. Details of the analysis and a schematic figure  on the eA manual review of prescriptions of all dispensed antibacterial products was performed retrospectively in 20 community pharmacies in the Southern Great Plain (SGP) region . The study period was between January and June 2007. Dispensed prescriptions of one workday (study-days) were reviewed for each month and for each pharmacy. The six study-days per pharmacy were selected by the double permutation method. Prescriptions pertaining to systemic antibacterials (ATC class J01) were identified. The dispensed quantity (number of sold boxes), patients’ ages, and product information  were recorded from prescriptions.All antibacterial products were grouped into three categories: parenteral, solid oral (e.g. tablet), and liquid oral (e.g. powder for suspensions). By capturing data on patients’ ages, the rate of antibiotics prescribed for children at different ages  could be calculated. The defined daily dose (DDD) content of products was assessed  to calculate the proportional use of liquid oral antibacterials . The association between these variables was analyzed by linear regression (SPSS 15.0). The strongest association was found between the ratio of liquid oral dosage forms and the age under 9 years . However, as the most common definition of ‘children’ is the age under 14 years, we used this age definition and the association with a slightly weaker correlation coefficient (R=0.898) for assessing pediatric antibiotic use. For comparative purposes, the weighted average DDD (defined daily dose) content of pediatric and adult prescriptions was also determined.For the same period, crude (i.e. number of dispensed packages) antibiotic use data for the whole region (N=445 pharmacies) was obtained from the National Health Fund Administration (NHFA). We converted data into DDDs . As in 2007, all antibiotics were reimbursed by the NHFA, all antibiotic sales in the region (covering 19.7% of the area and 13.3% of the population of Hungary) were included in the dataset. The ratio of liquid oral antibacterial use was calculated as described above. The result of the linear regression was applied to this aggregated dataset to estimate the rate of pediatric antibiotic prescriptions in the whole region. See We demonstrated that simple drug-related data (dosage form) can be linked to a patient-related measure (patient’s age). Based on this association, massive pediatric antibiotic exposure was found.This methodological approach could be applied when rapid general evaluation of prescription patterns are needed in simple aggregated datasets containing data in DDD format, like the European Surveillance of Antimicrobial Consumption (ESAC-net) database. Its use would also be feasible when a population-based prescription database is not in place or not available, does not contain age-linked data, or age stratification is impossible due to privacy/confidentiality issues.


In this paper, an all-fiber Mach-Zehnder interferometer (MZI) based on a non-zero dispersion-shifted fiber (NZ-DSF) is presented. The MZI was implemented by core-offset fusion splicing one section of a NZ-DSF fiber between two pieces of single mode fibers (SMFs). Here, the NZ-DSF core and cladding were used as the arms of the MZI, while the core-offset sections acted as optical fiber couplers. Thus, a MZI interference spectrum with a fringe contrast (FC) of about 20 dB was observed. Moreover, its response spectrum was experimentally characterized to the torsion parameter and a sensitivity of 0.070 nm/° was achieved. Finally, these MZIs can be implemented in a compact size and low cost. F [et al. . In addiIn this work, an all MZI based on a non-zero dispersion-shifted fiber (NZ-DSF) was experimentally demonstrated. We design our MZI by core offset fusion splicing of a segment of NZ-DSF between two SMFs. Moreover, the principal operation is discussed, as well as some experiments results and its spectral response characterization, is also provided.For the purpose of fabricating the MZI, two conventional single mode fibers (SMF-28) and a segment of a non-zero dispersion-shifted fiber (Corning Fiber Model SMFLF) were used. In ectively .In this way, in order to implement the MZI, both optical fibers were fixed into the splicer machine fiber holders (Fitel S175) and by manual mode, the NZ-DSF was displaced downward for a distance of 30 µm See, a. It is The principle of operation of this MZI can be explained as follows: when the fundamental mode travels through the SMF until it reaches the first core-offset joint See, , it is rThus, the phase difference for this type of MZI, among the core mode, ring core modes and cladding modes, can be expressed by 2π∆neL/λ , where ∆MZI arms . Hence, MZI arms .(1)∆λ=λOn the other hand, since the phase difference and separation fringes are wavelength dependent, the transmitted optical power, by the interferometer, will be present as a maximum at certain wavelength and a minimum at another . It is iIn order to characterize the MZI optical spectrum response, the experimental setup shown in L) were investigated. It is important to mention that the lateral offset of all these MZIs was 30 µm. The physical lengths of the NZ-DSF were L = 6.5, 5.5, 3.5 and 2.5 cm respectively. The output spectra using the lengths of 6.5 and 5.5 cm are shown in L = 2.5 cm, is higher than for other devices FC, implemented by using different techniques. For instance, it is around two times higher than at a MZI based on two cascaded long-period gratings (12 dB) [In this way, the output spectra of four MZIs with different physical length ( 12 dB) , and a M (12 dB) . Meanwhi (12 dB) ,17, or a dB , and (12 dB) .In order to determine the cladding modes and ring core modes that construct the interference spectrum, the fast Fourier transform (FFT) of the wavelength spectra is performed as is shown in A dip was done which is centered wavelength at 1504 nm  to apply the torsion effect See, . In this nm See, . Here, iMoreover, a shear stress along the circumferential direction occurred when the torsion is applied. As a result of the stress, the effective refractive indexes of the core, ring core and the cladding are changed . Since, Therefore, the change of the effective refractive index both for the core and ring core thickness are small, which means a growth on the effective refractive index of cladding. In this way, modifying the torsion effect can change the effective refractive index difference between cladding and ring core thickness and core r values . At thisWe have proposed and demonstrated a new Mach-Zehnder interferometer based on a non-zero dispersion shifted fiber. Here, the MZI was implemented by a core offset splicing of a NZ-DSF between two segments of SMF, and several fringe contrast of 2.25, 4.27, 5.22 and 20.54 dB were obtained respectively. Moreover, the MZI was tested for torsion sensing and it was observed that the torsion effect induced a nonlinear behavior due to the shearing stress that is not uniform at the core-offset MZI structure. Here, by experimental measurements, we determined a sensitivity of 0.070 nm/° to a physical length of the NZ-DSF of 2.5 cm.


The datawere collected using an interview script and physical examination. Logisticregression was used for the data analysis, considering a level of significance of10%.  the predictive factors identified were: for the nursing diagnosis of knowledgedeficit-inadequate following of instructions and verbalization of the problem; forthe nursing diagnosis of failure to adhere - years of study, behavior indicativeof failure to adhere, participation in the treatment and forgetfulness; for thenursing diagnosis of sexual dysfunction - family income, reduced frequency ofsexual practice, perceived deficit in sexual desire, perceived limitations imposedby the disease and altered body function.  the predictive factors for these nursing diagnoses involved sociodemographic andclinical characteristics, defining characteristics, and related factors, whichmust be taken into consideration during the assistance provided by the nurse. It is estimated that 35 million people are living with Human Immunodeficiency Virus(HIV). From the beginning of the AIDS epidemic (1980) through to December 2013,approximately 1.5 million deaths were identified which had Acquired Immune DeficiencySyndrome (AIDS) as their basic causeThe nurse is inserted in this context as an important facilitator of the care for peopleliving with AIDS, whether this involves undertaking the nursing procedures duringinpatient treatment or assessing and providing guidance in relation to their state ofhealth, tests, medications, diet or prevention of the transmission of the virus, amongother issues. For this, it is necessary for these actions to be undertakensystematically. The Systematization of Nursing Care can take place based on the application of theNursing Process, which is made up of five interrelated, interdependent and recurrentstages: data collection (the patient history), the nursing diagnosis, planning,implementation, and nursing evaluation. Through this, the nurse's practice is based onclinical judgment, greater organization, and quality of care. For this, it is essentialfor the professional to involve the people living with AIDS and their familymembersIn this respect, the identification of the nursing diagnoses and of their predictivefactors, which are related to the human responses, facilitates the implementation of thenursing interventions which will be most appropriate for the real necessities of thepeople living with AIDS - and for their socioeconomic and cultural context. Biblioteca Virtual em Saúde - BVS) were used: Latin Americanand Caribbean Center on Health Sciences Information (LILACS) and Medical LiteratureAnalysis and Retrieval System Online(MEDLINE); and Scopus and CINAHL ,using the following combinations: Nursingdiagnosis and Acquired Immune Deficiency Syndrome, Nursing process and Acquired ImmuneDeficiency Syndrome. It was ascertained that there were few studies addressing NursingDiagnoses in people living with AIDS-In order to support and justify the undertaking of this study, an integrative review wasundertaken, looking for scientific productions published in the last five years on theissue in question. For this, the following databases provided through the Virtual HealthLibrary ,sampling error of 5% and the size of the populationThe study included people diagnosed with AIDS, who were over 18 years old, and who hadbeen receiving inpatient treatment for at least 12 hours. The study excluded those whodid not have the physical or psychic conditions to participate in the study, as well asthose who were not aware of the diagnosis of the disease. It is emphasized that peopleliving with AIDS who were receiving inpatient treatment on a subsequent occasion, whohad already been interviewed in the present study, were not included again in thesample. The collection took place in the period March - September 2014, corresponding to thetime period necessary to reach the sample calculated. In approaching the subjects, theresearch proposal was presented, and the Terms of Free and Informed Consent were read.For this, the patients were given the time necessary for taking their decisions. If theywished to contribute to the study, a patient history was taken and the physicalexamination was undertaken in a separate room, in the above-mentioned hospital,respecting the patient's privacy. Throughout the data collection process, accessiblelanguage was used such that there would be no doubts or embarrassments.,-The instruments used in collecting the data, for taking the patient history and carryingout the physical examination were adaptedThe instruments were validated by eight professors with Ph.Ds with experience in nursingdiagnoses and Acquired Immune Deficiency Syndrome, who participated as judges. Theevaluation of the instrument took place based on the classification of each item inrelation to the judges' opinion regarding agreement or disagreement relating to the itemremaining in the instruments. Besides this, suggestions could also be made. Items whichreached a rate of agreement of ≥0.80 among the professors who participated in the studywere incorporated into the instruments, being considered validated. The above-mentionedinstruments were applied, in the form of a pretest, to 10 people living with AIDS, inthe study locale. There was no need for changes, and the participants were included inthe study's sample. The NANDA International, 2012-2014 versionA database was constructed which recorded all of the sociodemographic and clinicalvariables, the defining characteristics, the related factors and the nursing diagnosesidentified. The analysis of the data was undertaken descriptively, through obtainingabsolute and relative frequencies. For the numerical variables, the measurements ofcentral tendency were presented. Inferential statistics were calculated through logistic regression using the stepwisemethod in order to identify the predictors of the nursing diagnoses which influenced theprocess of establishing the human responses, presented by people living with AIDS; alevel of significance of 10% was considered. It is emphasized that it was impossible toundertake statistical tests for the nursing diagnosis of "ineffective protection", asthis presented a frequency of 100% among the participants in the present study. As a result, the logistic regression was undertaken for the three diagnoses mostprevalent in this study, with the sociodemographic and clinical variables, contained inthe research instrument , as well as the defining characteristics and relatedfactors, which presented a prevalence of 15%. The research project was approved by the Committee for Ethics in Research with HumanBeings, of the Federal University of Rio Grande do Norte (Opinion N. 508.445/2014),respecting the national and international rules for ethics in research involving humanbeings. The majority of people living with AIDS are male (n=82/72.6%), with a mean age of 39years old (±9.81), are heterosexual (n=81/71.7%), have studied for up to eight years(n=74/65.5%), have a family income of up to one minimum salary (n=66/58.5%), and live inthe interior - that is, the rural areas - of the State (n=60/67.8%). On average, thetime since diagnosis with AIDS was 5 years (±5.38), and 69 (n=78) had already abandonedtreatment through lack of belief (n=16/20.5%).A total of 56 nursing diagnoses was identified, with four being prevalent in more than50% of the people living with AIDS, these being: ineffective protection (n=113/100%),knowledge deficit (n=91/80.5%), failure to adhere (n=78/69%) and sexual dysfunction(n=61/54%).The frequencies of the defining characteristics of the Nursing Diagnosis (ND)of ineffective protection were: lack of immunity (n=113/100%), change in coagulation(n=76/67.2%), fatigue (n=70/61.9%), weakness (n=52/46%), and dyspnea (n=28/24.7%). Onthe other hand, for the related factors, those prevalent were: immunological disorders(n=113/100%), abnormal blood profiles (n=96/84.9%), drug abuse (n=51/45.1%), inadequatenutrition (n=25/22.1%) and drug therapy (n=16/14.1%). The predictive factors identified were: for the nursing diagnosis of knowledge deficit -inadequate following of instructions, and verbalization of the problem; for the nursingdiagnosis of failure to adhere - years of study, behavior indicative of a lack ofadherence, participation in the treatment and forgetfulness, and for the nursingdiagnosis of sexual dysfunction - family income, reduced frequency of sexual practice,perceived deficit in sexual desire, perceived limitations imposed by the disease, andaltered body function.-The AIDS epidemic, in Brazil, in recent years, has been marked by a change in theepidemiological profile, marked by heterosexualization, povertization andinteriorization ,-The nursing diagnosis of ineffective protection presented, in general, definingcharacteristics and related factors linked to the change in immunological status,including the changes of the blood cells. Studies undertaken previously corroborate thisfindingIt is known that HIV works by linking itself to the surface of the individual'simmunological cells, mainly destroying the CD4 cells, although it can also reach bloodcells, platelets and leukocytes, leading to a situation of fatigue, dyspnea, anemia,infections and hemorrhage. These hematological changes can also be somatized by theimmunosuppressive effect itself of the opportunistic infections and neoplasticinfections and by the myelotoxic effects which some antiretroviral medications, orcorticosteroid and antineoplastic medications, can cause-The literature also points to the presence of gastrointestinal changes as a predictivefactor for the worsening of the immunological situation, as diarrhea, in conjunctionwith the action itself of the virus, provokes change in the absorption of nutrients andthe metabolism of lipids, contributing to the appearance of a situation ofmalnutrition,Among the opportunistic infections and co-infections, emphasis is placed upon:pneumocystis, toxoplasmosis, tuberculosis, cryptococcal meningitis and infection bycitomegalovirus and herpes zoster, among othersCandida is observed with any level of CD4 Tcells, such that the reduction in these cells influences only its frequency andseveritySome of these infections are not related exclusively to the reduction in the CD4 Tcells, as in the cases of tuberculosis and candidiasis. The paradoxal reaction,resulting from the treatment of tuberculosis, can be caused by the liberation of theprotein from the bacillus during its destruction. This protein content may be understoodby the organism as an anti-gen, thus causing an exacerbated innate and adaptive immuneresponse. Infection by In the light of the above, the appearance of the disease's complications can be relatedto various factors, namely: mechanisms of the action of the virus, paradoxal effect ofthe treatment of tuberculosis, changes in the leukocyte cells and effects of theantiretroviral medications. These factors can lead to absence of interest in thetreatment, contributing to its abandonment. The nurse needs to monitor the clinical situation of the people living with AIDS, beingattentive to the blood markers and symptomatology, with an aim to identifying thecomplications caused by the disease. Furthermore, it is important for these people to beaware regarding their current clinical status and prognosis and, above all, regardinghow they can contribute to minimizing these. Regarding the nursing diagnosis of knowledge deficit, it is known that the inadequatefollowing of instructions, and verbalization of the problem, can influence the way thatpeople living with AIDS deal with the disease, as well as their adherence to thetreatment and to healthy behaviorsIn this regard, the nurse must encourage people living with AIDS to reports theirdoubts, sources of distress, and difficulties related to the treatment; principally atthe time when they find out about the disease, as it is common to present irritability,guilt or apathy, among other negative feelings, a fact which can negatively influencethese peoples' involvement for following the treatmentIt is understood that the more that people living with AIDS are informed, the morecapable they are for dealing with the situation, and the better they understand thatchanging their lifestyle and being committed to the treatment are crucial for a betterprognosis of the disease, and, consequently, for a good quality of life. Participation in the treatment can be significantly linked to the amount of informationwhich the individuals have regarding the disease, above all regarding the consequenceswhich irregularity in the treatment can cause, such as opportunistic infections andsystemic changes. The nurse can intervene in order to minimize the predictive factors for the nursingdiagnosis of knowledge deficit, through guidance regarding the disease, the prognosis,treatment and living habits which can favor the improvement of quality of life. It isalso necessary to take into account how this information will be passed on, principallyto those with a low educational level. -,In relation to the predictive factor of years of study, linked to the nursing diagnosisof failure to adhere, studies indicate that the low educational level of people livingwith AIDS predominates as an aspect which can influence lack of adherence to thetreatment of the diseaseIn one study undertaken in the United States of America, which aimed to analyze therelationship between the individuals' knowledge regarding HIV and their adherence to thetreatment, it was ascertained that knowledge on their state of health, including aspectsrelated to the disease's development, such as CD4 count and viral load, contribute tothe involvement of people living with AIDS in their adherence to the treatment,-Other factors are indicated by some studies as possibly influencing adherence to thetreatment: financial difficulties, nutritional factors, lack of belief in the treatment,drug use, depressive symptoms, the occurrence of adverse reactions during the taking ofmedications, and the quantity of antiretrovirals ingested per day-The financial difficulties limit access to appropriate food for minimizing the situationof physical debility, characterized by muscular weakness and low weight, resulting fromthe action of the virus itself, as well as the adverse reactions to the medications, orsymptoms of the opportunistic infections,Besides this, adverse events resulting from these medications can also involveanatomical, metabolic or neuropsychiatric changes, as well as gastrointestinal effectsof different intensities,It is known, however, that the antiretroviral medications have an important role in thecontrol, survival, and improvement of quality of life of people living with AIDS, asthey act in viral suppression and maintain the levels of the CD4 cells at a higherlevel, thus allowing an improvement in the immunological system and a reduction in theoccurrence of opportunistic infectionsIt is important to emphasize that, although in this study there was no statisticalsignificance between the diagnosis of failure to adhere and the variables related to theconsumption of alcoholic drinks, tobacco and illicit drugs, it is interesting to notethat the factors related to behavior which is indicative of lack of adherence andforgetfulness can result from this lifestyle, which is highly prevalent among theparticipants in this research.-,Studies confirm that unhealthy life behaviors, including the consumption of drugs, canbe determined both in contagion with HIV and in relation to adherence to the disease'streatment, as these people commonly - when ingesting alcohol or consuming other drugs -reduce their level of cognition and undertake specified behaviors, such as the practiceof unsafe sex, multiplicity of partners, the sharing of syringes, and forgetting to taketheir antiretroviral medicationsIn this regard, the nurse must be alert to the multiple factors which influenceadherence to the treatment, principally those referent to the behavioral questions.Involvement and the feeling of responsibility are fundamental for therapeutic success.Family and psychological support are also necessary in this process, in the sense ofencouraging people living with AIDS to follow the treatment appropriately, includingthose behavioral changes which have such an impact for a better prognosis of thedisease. It is necessary to involve these people in their health/illness process,principally by providing them with the possibility to choose, in conjunction with thehealth team, how the treatment will be carried out, also taking into account theextrinsic factors such as the socioeconomic and cultural factors. The nursing diagnosis of sexual dysfunction presented, as predictive factors, alteredbody function, family income, reduced frequency of sexual practice, perceived deficit insexual desire and perceived limitations imposed by the disease. One study indicates that great discomfort exists in relation to the bodily changescaused by AIDS and the antiretrovirals, mainly those related to the loss of weight andto lipodystrophy. This change in body image sometimes can cause insecurity or shame inpeople living with AIDS, being able to negatively influence their self-esteem and theirpersonal and sexual relationships, principally when this is related to a situation ofdepressionThe principal form of transmission of the disease can negatively influence the way thatpeople living with AIDS experience their sexuality, whether through fear of transmittingthe virus, or through associating sexual practice with their current health condition,mainly when these people present various episodes of inpatient treatment in a shortperiod of timeSexual dysfunction has multifactorial causes, which can be organic or psychological.Academics indicate that endothelial dysfunction can occur as a consequence of asituation of dyslipidemia, obesity, or diabetes mellitus, caused by the action of HIVitself, or by the adverse effects of the antiretrovirals. This process changes thevascular relaxation and blood flow, thus negatively influencing the sexual functioningof people living with AIDSHypogonadism has multifactorial pathogenesis, and can occur because of the cytopathiceffects of the virus in testicular cells, the use of gonadal toxic drugs, malnutrition,opportunistic infections and the neoplasias which affect the testicle. The low levels oftestosterone can be related to hypogonadism and can cause loss of muscle mass andstrength, reduction in bone mineral density, and lipodystrophy and sexualdysfunctionFinally, the nursing diagnosis of sexual dysfunction presented family income as one ofits predictive factors. Living with a disease which continues to be so stigmatized bysociety hinders the individual's access to the labor marketIn the light of the multi-causality which can involve the nursing diagnosis of sexualdysfunction, it is important for the nurse to establish a relationship of trust andrespect for the people living with AIDS for whom she provides care, so that she may cometo know the predictors which can lead to these dysfunctions occurring, or which havealready caused an impact on these peoples' quality of life. As contributions to the advance of scientific knowledge, it is emphasized that theidentification of predictive factors confers upon the nurse greater clinical power, asit allows the knowledge of those aspects which are related to the human responses ofpeople living with AIDS, and the extent to which these possess a multicausal dimensionand are interlinked. This identification facilitates the establishment of nursinginterventions which are more suited to these peoples' real needs, and to theirsocioeconomic and cultural context, as well as making it possible to minimize thecomplications of the treatment and the coexistence with the disease itself; also beingable to provide a basis for the teaching of the nursing diagnoses. The study's limits were related to the non-probabilistic type of sampling, which doesnot guarantee the sample's representativity. Another aspect identified consists of thefact that the clinical evaluation is a subjective process; in the light of this, thediagnostic process is subject to uncertainties. It is stressed that the small number ofstudies addressing the nursing diagnoses in people living with AIDS, and which usedinferential statistics, led to a certain difficulty for comparing the findings of thepresent study. In this regard, it is suggested that further studies should be undertaken with a view togiving a broader foundation for the nurse's care practice with people living with AIDS,principally in relation to the nursing diagnoses and their predictors. The study made it possible to identify that the predictive factors of the nursingdiagnoses of knowledge deficit, failure to adhere and sexual dysfunction, found in thepeople living with AIDS and involved sociodemographic and clinical characteristics,defining characteristics, and related factors, which can be used by nurses foridentifying the increased risk for the development of specific human responses andcomplications related to the disease.


Clostridium difficile infection (CDI) is an infectious diarrheal disease that is associated with antibiotic and healthcare exposures. Although individual-level risk factors have been extensively studied, the facility-level factors that drive CDI have not.To study the determinants of CDI incidence across long term care (LTC) facilities, with a specific interest in the role importation of infectious patients from acute care (AC) facilities.C. difficile lab-identified event, defined as a case with onset ≥ 3 days after admission occurring at least 8 weeks from a previous positive test.We conducted a retrospective cohort study of CDI from 2006 through 2012 across Veterans Affairs local healthcare systems (HCS) where both AC and LTC patient censuses were above an average of 10 patients per day. Our outcome was LTC-onset C. difficile infection in LTC facilities was 3.6 per 10,000 patient-days. In bivariate weighted linear regression analyses, the most important predictors of facility CDI incidence were importation  and antibiotic prescribing . Time-series analyses revealed that increases in C. difficile case importation from AC facilities preceded increases in CDI rates for a period of up to 8 weeks. Multi-level analyses, that included individual-level covariates, revealed that C. difficile importation and facility-level antibiotic use acted independently of resident age, direct antibiotic exposure and direct proton pump inhibitor use.We identified 90 local HCS that met our inclusion criteria. The incidence of C. difficile cases from AC facilities and facility-level antibiotic use are principal drivers of CDI in LTC facilities. A regional approach, addressing rates in AC facilities, is needed to control CDI in LTC facilities.This is the first study showing that importation of None declared.


Infections in orthopaedic surgery are a serious issue. Antibiotic-loaded bone cement was developed for the treatment of infected joint arthroplasties and for prophylaxes in total joint replacement in selected cases. Despite the widespread use of the antibiotic-loaded bone cement in orthopedics, many issues are still unclear or controversial: bacterial adhesion and antibiotic resistance, modification of mechanical properties which follows the addition of the antibiotic, factors influencing the release of the antibiotic from the cement and the role of the surface, the method for mixing the cement and the antibiotic, the choice and the effectiveness of the antibiotic, the combination of two or more antibiotics, and the toxicity. This review discusses all these topics, focusing on properties, merits, and defects of the antibiotic loaded cement. The final objective is to provide the orthopaedic surgeons clear and concise information for the correct choice of cement in their clinical practice. The purpose of this review is to analyze the main issues of antibiotic loaded bone cements and to comment their basic properties, main characteristics, merits, and defects. The final goal is to provide the orthopaedic surgeons of clear and concise information for a correct choice of antibiotic loaded cement in the clinical practice.The presence of biomaterials in orthopedic surgery involves a high risk of developing deep infections . One of The positioning into the surgical site of cement loaded with antibiotics may be useful to maintain at local level a high concentration of drug, which could not be reached by the venous administering without general complications and toxicity , 15. Nev The subject of this review, the so called bone cement is a polymer-based material composed of poly-methyl-methacrylate (PMMA) or copolymers, is a polymeric material commonly used for the fixation of the joint implants to the bone.  In recent years, thanks to the improved surgical techniques, to the adoption of stringent and efficient antiseptic pre-operative and intra-operative procedures and, above all, thanks to the optimization of the peri-operative systemic antibiotic prophylaxis a significant reduction in the number of deep infections and subsequent revisions occurred –20. It wS. Aureus would show preferential adhesion to metallic biomaterials [The addition of antibiotics to the cement must be considered as a support strategy in preventing the onset of infections and not the solution: the key point is still represented by the sterility in the operating room and by the antiseptic surgical procedures. Nevertheless, any procedure which can potentially reduce the adhesion and the bacterial colonization is welcome in orthopedics. Hypotheses were formulated about the effectiveness of the addition of antibiotics in terms of reduction of the bacterial biofilm on the different types of cement and it appeared as a multi-factorial process, not related only to the kinetics of release of the antibiotic. Some sustained that the production from the bacteria of a kind of glycocalyx , which adheres to the biomaterial, causes physiological changes on bacteria themselves and confers antibiotic resistance , 23. Othaterials . Prolongaterials , 22. Theaterials . Also, baterials , 29. Anoaterials .The PMMA enters the operating room packaged in monomer (liquid) and polymer (powder) separately. At the time of the preparation, when mixed, it becomes a viscous material paste, which solidifies in few minutes by an exothermic reaction. It acts as a fixation between the prosthetic components and the cancellous bone. During the mixing, pores of different sizes are produced as consequence of the chemical reaction and volume variations. These microholes may represent the start point of cracks and thus can be responsible of the premature failure of the cement. To avoid the formation of these pores, it is possible to prepare the cement under vacuum conditions. Nevertheless, the preparation under vacuum leads to a greater reduction in volume during the polymerization, thus resulting in higher shrinkage and worse adhesion on the bone-prosthesis interface compared to the nonvacuum mixed cement. It was demonstrated that among the compounds prepared in the operating room, those made under vacuum conditions present improved mechanical properties –32. Howein situ [Some authors do not recommend the use of antibiotic cement in primary arthroplasty , first oin situ , 30, 36.in situ .method of addition of the antibiotic to the cement: manually mixing at the time of implantation or industrial mixing by the several companies which provide premixed antibiotic loaded bone cement [The method of mixing is considered one of the most important factors that affect the release of the antibiotics and the mechanical properties of cement. The preparation should be as porous as possible in order to increase the spread of the antibiotic, but not excessively porous to weaken the structure of the cement itself. A fundamental distinction regards the e cement , 38, 39.The antibiotic must be a powder preparation for a better integration with the cement and a reduced interference with the mechanical properties of the cement . Until nStaphylococcus aureus). In addition, the antibiotic must provide a local concentration able to overcome the “break point sensitivity limit” of pathogens. This is generally defined as the antibiotic concentration that marks the transition from bacterial sensitivity to induction to resistance to antibiotics for at least three or four weeks. The final aim is to reach appropriate antibiotic concentrations in the tissues and bone avoiding the toxicity of the concentration of the drugs [The choice of the antibiotic is a fundamental issue. The antibiotic must have a broad antibacterial spectrum (including gram positive and gram negative bacteria) and a low percentage of resistant species. The most commonly mixed antibiotics are gentamicin and tobramycin (aminoglycosides with particular effectiveness against gram-negative bacteria) and vancomycin  compared to other cements. It was also noted that the gentamicin may act differently when added to different cements, although the mechanism of bacterial adhesion is always the same. For example, it was demonstrated that the release of gentamicin is much more effective in Palacos than in Simplex [A study showed that coagulase negative staphylococci are found in the 88% of the infections in patients undergoing a primary arthroplasty where cement loaded with gentamicin was used . A study Simplex . Similar Simplex . Other s Simplex . This lo Simplex . One study  showed tThe dosage of the antibiotic varies according to the use for which the cement is destined. Many authors argue that in case of acute infections high doses of antibiotics should be used: more than 2 g each 40 g of cement, usually from 6 to 8 g each 40 g, for a prolonged and effective release against pathogens , 22, 48.S. aureus). Since 1970, it has been documented that β-lactamic antibiotics can be combined with most of aminoglycosides, when there is a high concentration of both substances, or when their excretion is delayed. The combination of a molecule of a β-lactam with an aminoglycoside molecule can inactivate equimolar amounts of both antibiotics. When high doses of both substances are combined with cement, the inactivation of these can affect the properties of the combination, but this phenomenon has not been studied yet [The activity of the release of two or more antibiotics from the bone cement was studied. The idea to add more than one single antibiotic arose after the emergence of resistant bacteria and after the possible synergistic combination of two antibiotics has become an increasingly common practice in infectivology  were studied on hospital acquired staphylococcal strains systematically inoculated on four orthopedic materials: Ultra-High Molecular Weight Polyethylene (UHMPWE), Palamed cement without antibiotics, Palamed-G cement, and Palamed-G cement loaded with vancomycin (1 g of antibiotic each 40 g of cement) [A study conducted on 20 patients with infections due to in alone . The eme cement) . The sam cement) , 51.in vitro and may translate into enhanced elution in vivo [The additive or synergistic effect of tobramycin on vancomycin released from acrylic cement was demonstrated . This ph in vivo .μg/mL). Nevertheless, vancomycin resulted active for the first day. Considering the tobramycin, for groups with low and medium dose an inadequacy in the kinetics of release was observed again, but for the high-dose group  the duration of release was high. In general, Palacos has a much higher release level (above the MIC for most common pathogens encountered) and for a longer period of time compared to Simplex and tobramycin showed an improved efficacy profile compared to vancomycin [in vitro was measured with the purpose to establish the best pairing cement-antibiotic against specific bacteria . Palacos-R and three different antibiotics  were used alone or in combination (gentamicin plus vancomycin or teicoplanin plus gentamicin). The study showed that the combination of two antibiotics in a spacer has a bactericidal activity more prolonged than a spacer loaded with a single antibiotic. Also, the synergistic action of gentamicin and teicoplanin had superior bactericidal activity compared to gentamicin and vancomycin and the coupling of a glycopeptide with an aminoglycoside covers both Gram-negative and Gram-positive bacteria.The characteristics of elution of vancomycin and tobramycin alone and together were compared in two types of cement, Palacos and Simplex , dividedncomycin , 54. Howncomycin . In anotncomycin  the releAccording to some authors the release of the antibiotic can last for many days , while fThe release of the antibiotic from the cement is influenced by the type (viscosity) of the cement, by the surface of contact/exchange, by the conditions of the compound, and the type and amount of antibiotic. The antibiotic is released from the surface of the cement and from cracks and voids in the cement itself . The natβ-lactams, aminoglycosides, macrolides, and others investigating the susceptibility to antibiotics both from bacteria adherent to the cement and from nonadherent bacteria. The contact with the material gave significant differences in terms of growth for all tested antibiotics, with the exception of clindamycin. These data suggest that the characteristics of the surface of the material could be important in the interaction with bacteria and that the bone cement can lead to changes in bacterial adhesion to the biomaterial modifying the antibiotic resistance [The effect of the direct contact with the surface of biomaterials, such as PMMA, on the characteristics of the bacteria and a consequent possible change in the population and bacterial resistance were studied . Also, dsistance , 61.in vitro studies [It was suggested, as mentioned above, that the addition of antibiotics may play a role in weakening the structure and the mechanical properties of the cement. Various cements and antibiotics were compared, to determine which might be the more resistant over the time: studies conducted on Palacos-R, CMW1, and CMW3 with and without the addition of gentamicin or Simplex-P with erythromycin, colistin, or tobramycin did not show significant effects on fatigue resistance in comparison to the respective simple cements . It must studies . On the  studies , 49.in vitro studies raised doubts about this subject. In addition, the concerns are more consistent in case of cement loaded at high doses, where the local levels of antibiotics may exceed 200 μg/mL. In particular, when osteoblasts derived from trabecular bone were exposed to materials containing various concentrations of gentamicin (0 to 100 μg/mL), the activity of alkaline phosphates decreased significantly in all the cultures with gentamicin concentration >100 μg/mL, the incorporation of 3H-thymidine decreases at the same concentration of antibiotic, and the total DNA decreases for concentrations ≥700 μg/mL [μg/mL) on osteoblasts showed that local levels <200 μg/mL have no effect on replication of these cells, whereas at concentrations >400 μg/mL replication decreases, and with 10,000 μg/mL cell death occurs. Also the effects of vancomycin on osteoblasts were studied for concentrations ranging between 0 and 10,000 μg/mL: levels of vancomycin <1.000 μg/mL had little or no effect on replication, but concentrations of 10,000 μg/mL caused the death of the osteoblasts [At our knowledge, there are no reports in literature of systemic toxicity related to the use of ALBC. Various researches focused about local toxicity, with particular interest to the function of osteoblasts and osteocytes: event though there are no reports of clinical adverse effects on these cells, some 00 μg/mL , 63. A seoblasts . VancomyThe majority of the studies demonstrated an antibacterial effectiveness of cements loaded with antibiotics in the treatment of deep infections following hip and knee arthroplasty (onestage and two stages). in vitro showed that the highest concentration of antibiotic released is found in the first two days; in contrast, studies in vivo did not reach statistically significant evidence.The main problem of these analyses is the minimus time of treatment that allows the antibiotic effects without developing bacterial-resistance. Recent studies The literature demonstrated that the best results are obtained with the association of antibiotic-loaded cement and the systemic antibiotic administration, if possible with targeted testing.Finally, a significant difference between the intravenous administration of antibiotics and the use of the antibiotics into the cement for prophylactic use in patients with standard risk was not found; so it is not advisable to use antibiotic loaded cement for routine as prophylaxis.  In conclusion, it is recommended not to trust excessively in the role of antibiotic loaded bone cement and not to give it therapeutic properties that it does not posses. It is clear how ALBCs are more effective than simple cements, but undoubtedly the “window of effectiveness” cannot be attributed only to antibiotics. Other properties related to the cement itself such as roughness, porosity, technique of preparation, and many patient-related features must be reminded. However, it is necessary to underline that ALBC, especially if targeted by a specific antibiogram or integrated with an association of molecules more than a single one, is an important aid in the prevention and in the treatment of prosthetic infections.