C. elegans nematode development. The devices can integrate PDMS layers and allow for temporal control of nutrient conditions and the presence of drugs on a minute timescale. We show how spatial confinement of motile C. elegans enables for time-lapse microscopy in a parallel fashion.The ability to spatially confine living cells or small organisms while dynamically controlling their aqueous environment is important for a host of microscopy applications. Here, we show how polyacrylamide layers can be patterned to construct simple microfluidic devices for this purpose. We find that polyacrylamide gels can be molded like PDMS into micron-scale structures that can enclose organisms, while being permeable to liquids, and transparent to allow for microscopic observation. We present a range of chemostat-like devices to observe bacterial and yeast growth, and  The ability to create precisely controlled microenvironments has been pursued in microbiology , cell biin situ valves [PDMS-based microfluidic devices are extremely versatile and have been applied to the culture of bacteria , yeast , mammaliu valves . Watertiu valves , which cu valves . PDMS isu valves , which iu valves ,18 and au valves . In addiu valves ,21.Many of these issues could be addressed by the use of hydrogels. Hydrogels allow for free diffusion of the medium throughout the device, thereby ensuring uniformity of the cellular environment. In addition, hydrogels have highly tunable mechanical properties . For theG ~ 10-50 J.m-2 [G ~ 0.1-6 J.m-2 [in situ microfabrication [Here we propose polyacrylamide hydrogels as an alternative substrate for building controlled micro-environments. Polyacrylamide gels have several practical advantages that make them ideally suited for developing devices for live microscopy in biological studies. First, polyacrylamide is a commonly used material in biology laboratories for DNA and protein electrophoresis and its microfabrication requires minimal technological investments, as we will show below. Polyacrylamide gels are physico-chemically well-characterized and known to be biocompatible ,37. They50 J.m-2 ) than ag-6 J.m-2 ,40) and -6 J.m-2 ,41,42. Trication , to builrication ,45 or inrication , their uE. coli bacterial colonies by controlling the carbon source as a function of time, and simultaneously track the response on the level of single-cell lineages. We also show that we can confine and grow S. pombe yeast cells, imposing a time-controlled reversible depolymerization of microtubules by a drug. Finally, we show that we can confine C. elegans nematode larvae in microchambers and follow the growth of multiple individual larvae in parallel by time-lapse microscopy.Here, we demonstrate two essential properties of polyacrylamide gels that enable the building of controlled environments and can be used simultaneously. First, we describe a soft lithography method to transfer micropatterns, such as confining culture chambers and microchannels, from a silicon wafer to a polyacrylamide gel. Second, we show how to use polyacrylamide gels as membranes to control the transfer of chemicals, building on previous designs using other materials that use dialysis membranes to change medium in time  or that Microfabricated polyacrylamide membranes can be indefinitely reproduced by soft-lithography from a single silicon wafer comprising the desired micropattern made of an epoxy resin, such as SU-8 (Methods). The initial master mold was made according to standard protocol by UV-lithography from a printed transparent mask, allowing the specification of an arbitrary two-dimensional pattern with a uniform height determined by the user, typically ranging from 1 to 100 microns.The aqueous solution of acrylamide monomers mixed with curing agents is poured on the master mold within a contour made of glass or metal of pre-determined height, bound to the wafer with silicon grease Methods, . The molAfter polymerization, the top silanized coverslip is removed, after which the gel is cut to the experimentally required dimensions and removed with tweezers. Importantly, the polyacrylamide membranes should be rinsed in water to remove unpolymerized acrylamide monomers, which are in particular known for their neurotoxicity. Proper rinsing has already been demonstrated to ensure biocompatibility of polyacrylamide matrices for neuron growth and development for weeks . TransfeIn -1 allow one to switch the media within seconds, generating pressures below 100 Pa, allowing continuous use without leakage for at least a day. As the mechanical clamping used in all these designs avoids irreversible sealing or chemical bonding, the PDMS channel can be re-used many times.In all different designs the objects under study are confined between the polyacrylamide membrane and the glass coverslip through which microscopy imaging is performed. Here, we use two different approaches to achieve this. In the first design, we enclose the membrane within a surrounding glass spacer sealed to a top glass slide with vacuum silicon grease . This siPotentially, microfabricated polyacrylamide membranes and PDMS control channels can be combined depending on the experimental needs . PossiblFirst, we used the advantageous transport properties of polyacrylamide gels to precisely control the medium composition in time, using an unstructured gel as a membrane, as well as in space, by setting up a concentration gradient within a membrane comprising molded microchannels.-1. A fluorescent glucose analog (2-NBDG) was added or removed from the flowing medium at a particular time point by switching a valve, thereby changing the composition of the flowing medium within seconds. The amount of 2-NBDG fluorescence was measured as a function of time by standard fluorescence microscopy using a 100X objective focused on the gel-glass interface. After the change of medium, the measured fluorescence signal rose in an approximately exponential fashion to the newly imposed steady-state value with a half-time of ~5 min .In the first experiment, time control of the medium was obtained by placing a 500 µm thick gel membrane between a structured PDMS layer and a glass coverslip . Flow waf ~5 min . Fits to-1. One channel contained pure water, whereas the other contained an aqueous solution of fluorescein molecules. Diffusion of fluorescein into the polyacrylamide hydrogel, coupled with its removal at the adjacent channel, gave rise to a linear concentration gradient in the space between the two channels [In the second experiment, we aimed to set up a spatial concentration gradient by placing a structured gel membrane between a glass slide and a flat PDMS layer, the latter containing inlet and outlet connectors . Liquid channels . We founchannels .E. coli bacteria . The lac genes control the import and catabolism of lactose and are induced during growth on lactose, but repressed when glucose is present.To show the ability of our designs to study cell dynamics in changing environments, we performed a carbon shift  systemaWe have tested various device-designs which allow accurate spatio-temporal control of the environment. Control in time was achieved by diffusion of the medium through the permeable gel into culture chambers that are hence uncoupled from the medium flow. Diffusion coefficients of small molecules in the gel are close to their value in water, which combined with the possibility to build thin membranes resulted in a medium exchange with a characteristic time of 5 min. This response time is well-suited for many applications that require the control of the growth and gene expression dynamics in micro-organisms such as bacteria or yeast. We demonstrated the creation of linear concentration gradients between two continuously flowing solutions in channels embedded in a single gel layer. This method may be used to set up gradients that are steeper or have more complex two-dimensional patterns than flow-cells used so far. In these existing designs, diffusion between PMDS channels is indeed constrained to occur through an unstructured agarose layer in a third dimension . We haveWe found that bacteria and yeast grew in well-defined monolayers when covered with polyacrylamide, as is also observed for agar pads, thus allowing convenient microscopy and analysis at the single cell level. The soft confinement provided by the polyacrylamide membranes ensured localization of the colonies, while maintaining normal growth and morphological phenotypes without requiring the deposition of an additional soft layer on the glass . In our C. elegans. Polyacrylamide gels with microchambers provide important advantages. First, they allow spatial confinement of these otherwise highly motile organisms, enabling time-lapse microscopy and parallel image acquisition without the use of anesthetic drugs [The technology is also promising for the study of larger, multicellular organisms, such as ic drugs  or automic drugs . Second,Finally, the tunable mechanical properties of polyacrylamide hydrogels make them potentially useful for the culture of other cell types, given for example the exquisite sensitivity of mammalian cells to the mechanical properties of their support . The potMaster molds have been realized on silicon wafers with spin-coated SU-8 epoxy resins (MicroChem) of different viscosities  resulting in heights of 3 µm for the yeast chambers, 18 µm for the worm chambers and 100 µm for the gradient assay. No specific wetting treatment was done to the surface of the wafer. For the polyacrylamide gel, we used a 29:1 ratio of acrylamide / bis-acrylamide (Bio-Rad) with a final concentration of 10%. Polymerization was initiated by the addition of 0.1% of ammonium persulfate (Sigma) and 0.1% of TEMED (Sigma). The mixture was poured in a mold consisting of a cavity made of a machined glass or aluminum slide of thickness varying between 150 µm and 1 mm, glued to the wafer or to a simple silanized glass slide with vacuum silicon grease. A silanized glass coverslip was deposited on top and the solution was left to polymerize for about 2 h. The polyacrylamide membrane was then cut and transferred in DI water for conservation. The PDMS (Dow Corning) channel was molded on a silicon wafer with SU-8 according to the protocol provided by the resin manufacturer (MicroChem) and consisted of a 113 µm high and 5 mm wide channel comprising pillars to ensure uniformity of the pressure applied on the polyacrylamide membrane. Mechanical clamping of the whole device was ensured by a home-made metal holder with 4 screws, comprising openings on the bottom for microscopy acquisition and on the top for illumination and the tubing. To ensure a good seal between the polyacrylamide and the PDMS layer, we made sure that the hydrogel and the PDMS had a contact area that extended at least 3 mm away from the edges of the control channel.The flow was externally driven with syringe pumps  connected to the microfluidics device by polyethylene tubing of 0.58 mm internal diameter . When using a PDMS channel, the device was degased 1 h in low vacuum prior to flow to allow removal of trapped air bubbles by suction from the PDMS matrix. Switches were performed by a manual valve . All experiments have been performed with an inverted epifluorescence Nikon microscope TE-2000 U embedded in a temperature controlled box.E. coli MG1655 (rph-1 ilvG- rfb-50). To measure the expression of the lac operon, lacA was replaced with GFPmut2 . Cells were grown in M9 minimal medium  with 0.2 mM uracil, supplemented with 0.1% (w/v) lactose, maltose or lactate and 0.001% (w/v) lactose in the limiting case. Note that adding uracil compensates for intrinsic pyrimidine starvation of the MG1655 strain [& al[-5 volume fraction) allowed further enhancement of colony growth into a monolayer. All these steps and the experiment were performed at 37°C. Images were acquired with a 100X oil immersion objective . Phase contrast and fluorescence images were analyzed with a custom Matlab algorithm derived from an algorithm of the Elowitz lab  [Growth experiments were performed using derivatives of 5 strain  and accoain [& al. . CellsCaltech) . The insS. pombe wild type fission yeast PT286 h- ade6-M216 leu1-32 ura4-D18. For the pad growth experiment, fission yeast cells were pre-grown overnight in Edinburgh Minimal Medium (EMM) liquid culture. 2 µL of 10x-concentrated culture were deposited on a polyacrylamide gel membrane that had been incubated in EMM medium. All these steps and the experiment were performed at 32°C. Cells were imaged through a 40X objective oil immersion lens . Colony area was measured with ImageJ (http://rsbweb.nih.gov/ij) software. For the drug shift experiment, the same culture conditions were used for a strain expressing GFP tubulin (DB 871 h90 nmt1-GFP-tub:lys1+ leu- ura-) and microtubule detection was performed visually. For the confined growth experiment, we used a YE5S medium and performed imaging with a 20X objective  at 37°C.For growth experiments, we used the C. elegans strain was grown on NGM agar plates covered with E. coli OP 50 as food source, following standard protocols [The wild-type (N2) rotocols . Before rotocols , were trFormula was taken from Crank :l = 500 µm (width of system), x = 0 µm (position of measurement), C is concentration and t is time, values taken from N = 3. The maximum concentration C0 and the diffusion coefficient D were used as fitting parameters.where Figure S1Molding of the polyacrylamide gel and assembly of the device.A) Step 1: a cavity is prepared, consisting in the silicon wafer with the photoresist pattern, reversibly assembled with vacuum grease to a glass or metal contour of desired height. Step 2: the acrylamide solution is injected with a pipette within the cavity. Step 3: A silanized coverslip is then added on top of the cavity and polymerization occurs at room temperature for 2 h. B) Photographs of a multi-layered device: separate parts (top) and assembled device (bottom).(TIF)Click here for additional data file.Figure S2SU-8 pattern on a wafer and molded acrylamide gel. A) Image of a silicon wafer with 3 µm high patterns in SU-8 photoresist. B) Structures shown in A have been molded in a polyacrylamide gel. Scale bars 100 µm. The smallest features are 10 µm wide.(TIF)Click here for additional data file.Figure S3Growth rates in batch cultures.Optical density at 550 nm normalized by the OD at t=0 (and shifted for clarity) versus time for batch cultures of MG1655 cells growing in minimal medium with abundant (0.1%) glucose (dotted line), lactose (green), maltose (red) and lactate (yellow) as sole carbon source. Exponential fits to the experimental data points (lines) yielded growth rates of 1.12 h-1 on glucose, 1.01 h-1 on lactose, 0.88 h-1 on maltose and 0.54 h-1 on lactate, comparable to those obtained for cells growing in the microfluidic device .(TIF)Click here for additional data file.Movie S1(AVI)Click here for additional data file.Movie S2(AVI)Click here for additional data file.Movie S3(AVI)Click here for additional data file.Movie S4(AVI)Click here for additional data file.


This report presents a surgical technique to remove a broken cannulated nail from the femur. A Harrington rod was modified for retrograde impaction of the retained fragment. The broken implant was finally removed without complication. This particular procedure was safe, simple, and promising. Removal of a broken femoral nail especially a retained distal fragment is a challenging procedure in orthopaedic surgery. Generally, pulling of the broken distal fragment is determined as a primary procedure for removal; however, slippage of the extraction device remains a major course of failure. Retrograde impaction is another promising method to overcome such problem. In this particular situation, an appropriate impactor is a key factor of success. Normally, another intramedullary nail or an extraction rod can be applied for such instance . HoweverA 35-year-old Thai man underwent closed femoral nailing as a treatment of right femoral shaft fracture. The implant was 11 × 38 mm AO interlocking nail for the femur. Six months later, he sustained another road traffic accident and presented with deformity and pain at the right thigh. The radiographs revealed distal 1/3 femoral shaft fracture with displacement and retained broken femoral nail Figures . TherefoThe patient was placed supine on a fracture operating table with pulling the affected leg on a skeletal traction at the proximal tibia . The proThe broken nail could be removed successfully followed by nail exchange without complication. No significant knee pain or functional limitation has been complained.Broken femoral nail removal is recently not an uncommon procedure in orthopaedic surgery. In general, the proximal portion of a broken nail is routinely removed without difficulty while challenge remains in the part of distal segment removal. For this instance, closed technique has been usually attempted as a primary procedure in order to preserve the surrounding soft tissue. Therefore, many surgical techniques have been published in the literatures to achieve such purpose –4. For broken cannulated nails, the distal portion can be removed by pulling or pushing of the retained implant from either the proximal or distal femoral canal. Normally, pulling technique is determined primarily because it is sequential following proximal nail removal and knee arthrotomy is not required. Many instruments such as hook, femoral head cork screw, smaller nail, multiple guide wires, and guide wire with washer have been recommended to be used as an extractor , 5–10. AFor retrograde impaction, size and length of the impactor remain a concerning issue in order to perform its function and compromise the risk of knee injury. Ordinary bone impactor is too short to be used for such instance. We found that Harrington rod can be perfectly modified as an impactor for this particular condition. Diameter of the rod is just only 6 mm; the round-end tip can engage into the broken nail lumen while the rod shoulder can hit against the nail tip allowing a mechanical benefit during impaction and low risk of slippage. The maximal working length of Harrington rod in our practice, tip-shoulder distance, is 38 mm which is suitable for retrograde impaction of the broken nail at the level of midshaft or distal 1/3. This method is simple, safe and no serious complication occurs in our experience.


The good use of antibiotics stays the best means to protect available ones. Our work aims to estimate the practices of prescription of antibiotics according to the national recommendations in a reference department.A descriptive and analytical prospective audit was conducted from April 2nd till July 2nd, 2012 from all cases of hospitalized patients having benefited from an antibiotic treatment with curative aim.The study concerned 170 patients with a total of 267 prescriptions of antibiotics. The average age was 41 years [15-86 years] and the sex ratio 1.5. The majority of the prescriptions were issued by -doctors in specialization (61.8 %) and- students in the last year of medicine (37.6 %). The main listed infections were: pneumonia (28.3 %), meningitis (21.80 %), opportunist infections during HIV infection (14.70 %) and tetanus (13.52 %). From the point of view of the prescription, 57.70 % of the patients had received a monotherapy: essentially ß-lactames (55.43 %), imidazoles (16.5 %) and macrolides (13.9 %). The most commonly used antibiotic combinations were: Amoxicillin-acid clavulanique-spiramycin (37.66 %), ceftriaxone-metronidazole (11.7 %), ceftriaxone-metronidazole-gentamicin (10.4 %). The rate of conformity of the antibiotic prescription with the national recommendations was 74.7 % in particular for the posology (92.50 %), the frequency of administration (95.88 %), the duration of treatment (55.43 %). Only 15.9 % of antibiotic treatments were re-evaluated at the end of 48 hours with a modification of the antibiotic regimen in 60 patients (35.5 %), in particular a broadening of spectrum (83.33 %). The average length of stay was of 14±11 days and mortality was 69.18 %.The rational use of antibiotics is essential to protect available drugs. It would be necessary to train the physicians and to advertise the user guide of antibiotics to improve the quality of the prescription.None declared


This study aims to determine antibiotic usage rates, causes of antibiotic usage and inappropriate usage rates in a university hospital with a 1,200-bed capacity.The study assessed antimicrobial drug usage among all hospitalised patients in the hospital on April 20, 2012, using the point prevalence method. Data were recorded using pre-prepared forms. Appropriateness of antibiotic usage was determined according to the appropriateness for the cause of antibiotic usage, the spectrum of the chosen antibiotics, usage dose, dose frequency and time of usage.Of 666 patients staying in the hospital on the day of study, 262 (39.7%) were on antibiotics. Of those, 145 (55.3%) were on surgical wards, 98 (37.4%) were on medical wards and 19 (7.3%) were on paediatric wards. Of those 262 patients, 157 (59.9%) were taking only one type of antibiotic, 79 (30.2%) were taking two and 26 (9.9%) were taking three or more types of antibiotic. Antibiotic usage was appropriate in 55.7% and inappropriate in 44.3%. The inappropriate antibiotic usage rate was 75.9% among patients on surgical wards. The most common cause of inappropriate usage was unnecessarily long prophylaxis time (68.2%). Inappropriate antibiotic usage was found in 24 (24.5%) patients out of 98 patients on medical wards. When the causes of antibiotic usage were analysed, it was found that the cause of antibiotic usage was infection in 36.2%, prophylactic in 35.9%, and empirical in 27.9%. On the day the study was conducted, 367 antimicrobial drug were prescribed to 262 patients. The drugs most commonly prescribed were antibiotics from the cephalosporin (27.0%) and fluoroquinolone (20.2%) groups. When the diagnosis of 95 patients who were on antibiotics due to infection was reviewed, the most common infections were respiratory tract infections (37.9%), urinary system infections (12.6%), upper respiratory tract infections (8.4%), bloodstream infections (6.3%) and prosthesis infections (6.3%).Inappropriate antibiotic usage rates in surgical wards were high. This inappropriate usage is especially related to prophylaxis time. This is why it is necessary for surgeons to be educated regarding prophylactic antibiotic usage and to stick to the surgical prophylaxis guidelines.None declared


Ufo1 complex for ubiquitylation. Both ubiquitylated Ho and Ufo1 are transferred by the UbL-UbA protein, Ddi1, to the 19S Regulatory Particle (RP) of the proteasome for degradation. The Ddi1-UbL domain binds Rpn1 of the 19S RP, the Ddi1-UbA domain binds ubiquitin chains on the degradation substrate. Here we used complex reconstitution in vitro to identify stages in the transfer of Ho and Ufo1 from the SCFUfo1 complex to the proteasome. We report SCFUfo1 complex at the proteasome formed in the presence of Ho. Subsequently Ddi1 is recruited to this complex by interaction between the Ddi1-UbL domain and Ufo1. The core of Ddi1 binds both Ufo1 and Rpn1; this interaction confers specificity of SCFUfo1 for Ddi1. The substrate-shield model predicts that Ho would protect Ufo1 from degradation and we find that Ddi1 binds Ho, Ufo1, and Rpn1 simultaneously forming a complex for transfer of Ho to the 19S RP. In contrast, in the absence of Ho, Rpn1 displaces Ufo1 from Ddi1 indicating a higher affinity of the Ddi1-UbL for the 19S RP. However, at high Rpn1 levels there is synergistic binding of Ufo1 to Ddi1 that is dependent on the Ddi1-UbA domain. Our interpretation is that in the absence of substrate, the Ddi1-UbL binds Rpn1 while the Ddi1-UbA binds ubiquitin chains on Ufo1. This would promote degradation of Ufo1 and disassembly of SCFUfo1 complexes.The F-box protein, Ufo1, recruits Ho endonuclease to the SCF The Ubiquitin-proteasome system has a major role in regulation of cellular processes, in particular the cell cycle and many signaling pathways UbL-UbA proteins are often referred to as shuttle proteins based on their recruitment of the ubiquitylated substrate from the E2-E3 complex and transfer to the 19S RP. This is supported particularly by the interaction between Rad23 and Dsk2 with the chain elongating E4, Ufd2, that occurs in the framework of a complex between Ufd2 and the AAA-ATPase ring hexamer, Cdc48 S. cerevisiae Cdc53, with the RING protein, Rbx1, attached to a C-terminal domain S. pombe Pop1-Pop2 FBPs The SCF complex comprises a rigid cullin scaffold, in ddi1Δ mutants ubiquitylated Ho endonuclease accumulates in the cytoplasm and is not transferred to the proteasome for degradation rpn1-D517A mutation that disrupts binding of Ddi1 to the proteasome stabilizes Ufo1 Ddi1 is required for the final stages of proteasomal degradation of both Ho endonuclease UFO1 has no obvious phenotype under normal growth conditions, however, a genomic UFO1 allele deleted for the UIMs is dominant lethal. Ectopic high level expression of UFO1 without the UIMs leads to stabilization of the protein and to cell cycle arrest at the end of G1. Substrates of other FBPs accumulate suggesting that Ddi1 is required for disassembly of SCFUfo1 complexes and recycling of the core complex subunits into alternative SCF complexes A protein fragment comprising the Ufo1-UIMs interacts with all three UbL-UbA proteins, Rad23, Dsk2, and Ddi1, however full-length (FL) Ufo1 interacts only with Ddi1 suggesting that the core residues are important for specificity in vitro to augment our in vivo data showing a role for Ddi1 in degradation of Ho and of its cognate FBP, Ufo1 Ufo1 complex to the 19S RP and subsequent degradation of Ufo1. We delineate stages in the formation of SCFUfo1-Ho-Ddi1-19S RP complex. Domain analysis showing different modes of interaction of Ddi1 with Ufo1 and Rpn1 in the presence and absence of Ho support the “Substrate shield” model of protein degradation Here we used complex reconstitution GFPFL-Ufo1, or Ufo1 truncated for the C-terminal UIMs, GFPUfo1Δuims, with GSH beads bound to recombinant GSTFL-Ufo1, GSTUfo1-WD40 domain, GSTUfo1-UIMs, or control GST . In contrast addition of yeast extract with ubiquitylated GFPHo to the reaction mix with either GSTUfo1 or GSTRpn1 did not affect the binding of either protein to HISDdi1 (Lanes 8 and 9). Furthermore, Ho in the reaction mix comprising Ufo1, Rpn1, and Ddi1, abrogated the competition between Ufo1 and Rpn1 and all three proteins bound the HISDdi1 beads (Lane 10) and The Ddi1-UbL domain binds both the Ufo1-UIMs and Rpn1 GSTUfo1 from binding to HISDdi1 by GSTRpn1 is concentration dependent. We calibrated the system by determining an amount for each lysate/extract that would give detectable binding of protein to the Ddi1 beads . To address this question we repeated the synergistic binding experiment described in  of Rpn1 .in vitro indicated that SCFUfo1 complexes that contain their substrate, Ho, are associated with the 19S RP. These complexes can assemble in the absence of Ddi1, however, in experiments with extracts from w.t. cells Ddi1 is found in association with the SCFUfo1-Ho-19S RP complex. Our interpretation is that Ddi1 is recruited to preformed SCFUfo1-Ho-19S RP complex. Based on our previous experiments in vivo we propose that Ddi1 enters the SCFUfo1-Ho-19S RP complex via initial interaction between the Ufo1-UIMs and the Ddi1-UbL  we observed two different modes of interaction of Ufo1 and Rpn1 with Ddi1: (a) competitive - Rpn1 excludes Ufo1 from binding to Ddi1; (b) synergistic - high levels of Rpn1 formed a tertiary complex between Ufo1, Ddi1, and Rpn1. The competitive interaction indicates that the Ddi1-UbL has higher affinity for Rpn1 than for the Ufo1-UIMs. The dependence of synergistic binding of Ufo1 and Rpn1 on the Ddi1-UbA domain suggests that Ub chains on Ufo1 are involved. The higher ratio of Rpn1 to Ufo1 in the in vitro Ddi1 synergistic binding experiment could parallel molecular crowding within the SCFUfo1-19S RP complex. Thus in the absence of Ho our data support a complex in which the Ddi1-UbL is bound to Rpn1 while the Ddi1-UbA domain binds Ub chains on Ufo1. This model for sequential transfer of Ho and of Ufo1 to the 19S RP is presented in The “substrate shield” model proposes that the substrate protects the FBP from degradation MATa; his3Δ1; leu2Δ0; met15 Δ0; ura3Δ0), and ddi1Δ  were purchased from Euroscarf. Strains with genomic RPN1-GFP and RPN11-GFP are from the library of Wild type BY4741 , dcm, gal, lacY1, pRARE  (CmR) were used for most recombinant protein expression. His-tagged recombinant proteins were expressed in BL21 (Promega) , dcm, gal, λ(DE3), pLysS (Cmr)) or M15 (Qiagen) ) bacteria.XL1 MRF1 (StrataGene)  gyrA96 end A1 Δβ(lacz)M15/recA1 laclq proA+B+ lacF’::Tn10 relA1 supE44 thi hsdR17(rk-mk+) was used for plasmid amplification.Rosetta bacteria (Novagen)  are described in MT2989 for expression of MYC-CDC53 from the GAL promoter was obtained from M. Tyers YE-RPN11-GFP in which expression of RPN11-GFP is from the ADH1 promoter was obtained from M. Glickman Growth media and yeast transformation by LiOAc are as in HB2-GST-CDC53, pGST-DDI1, pGST-DDI1-ΔUBL, pGST-DDI1-ΔUBA, pHIS-DDI1, pHIS-ΔΔDDI1, pGST-RPN1, and pGST-RPN10 (gift of Dorota Skowyra); pGEX-5X-1 (Amersham Biosciences) was used to construct pGST-UFO1 by amplifying the UFO1 gene from genomic DNA using primer pair Ufo1F (GAATTCATGGAGCGGCCTGGCTTGGTATT) and Ufo1R (CTCGAGTCAATTGATTTCACTCAATGACAACG). pGST-UIMs was constructed using Ufo1UIMsF (GAATTCAAAACGACATTCAGTTGAGAATTGCA) and Ufo1R. pGST-WD40 employed primer pair WD40GstF (GAATTCAATATTAATGCTGCAGTG) and WD40GstR (CTCGAGGTTTTCTTCATCGGTGTC). pCDFduet1- HIS-UFO1 was constructed by amplifying the UFO1 ORF with primer pair Ufo1HisF (GGATTCATGG AGCGGCCTGGCTTGGTATT) and Ufo1HisR (CTCGAGTCAATTGATTTCACTCAATG ACAACG). pCDFduet1- HIS-WD40 was constructed by amplifying the WD40 domain of UFO1 with primer pair WD40HisF (GGCGCGCCAATATTAATGCTGCAGTG) and WD40HisR (CTCGAGGTTTTCTTCATCGGTGTC).pImmunoprecipitation and immunoblotting were performed as described in GAL promoter by overnight growth in minimal medium with 2% galactose. Next morning the culture was diluted 1∶3 and grown for a further 1.5 hours. 50 mls of logarithmic culture served as the source of a 300 µl extract with 80 µg/µl protein. 200 µl were taken for immunoprecipitation (IP) with the appropriate antibody and the immunoprecipitate was run in a single lane for Western blotting (WB).Mouse anti-GFP (Roche Applied Science), mouse 9E10 anti-myc (Enzo), and mouse anti-HIS (Sigma) antibodies were used at a dilution of 1∶250 for IP and at 1∶1,000 for WB; mouse anti-GST (Santa Cruz Biotechnology) antibodies were diluted 1∶1,000 for IP and 1∶2,000 for WB, rabbit anti-Ddi1 (gift from Jeffrey Gerst) was used at 1∶5,000 for WB. Goat anti-mouse and anti-rabbit antisera, used at 1∶1,000 were from Santa Cruz Biotechnology. Protein A-sepharose was purchased from Amersham and used at 50%; 30 µl were added to each sample.TCA precipitation proteins were precipitated from 300 µl cell extract by adding TCA to 10% with 10 minutes incubation on ice. The pellet was centrifuged at 12,000 g for 10 minutes and five volumes of cold acetone were added. The protein pellets were harvested and dried. For WB analysis the pellets were dissolved in 30 µl of sample buffer and 5 µl of each fraction was separated by SDS-PAGE.µg/ml. A single colony was grown in 1 liter of LB (with ampicillin and chloramphenicol) to an OD600 of 0.6–0.8 (3–5 hours) with vigorous agitation at 37°C. IPTG was added to 0.4 mM to induce expression and the culture was incubated overnight at 20°C. The cells were harvested by centrifugation at 4°C for 10 min at 6,000 rpm. The cell pellet was washed with 20 ml of ice-cold PBS and resuspended in 3 ml yeast extract buffer ). The cell suspension was disrupted with an ultra-sound sonicator on ice using 6 cycles each of 10 seconds and clarified by centrifugation for 10 min at 4,000 rpm at 4°C. The supernatants with the GST-fusion proteins were incubated with Glutathione-sepharose 4B (GSH) beads (Amersham Biosciences) prewashed in yeast extract buffer with 1% Triton-X100; HIS-fusion proteins were incubated with washed Ni-sepharose (Clontech) for 1.5 hr at 4°C. The bead fractions were washed 5 times in extract buffer with 2.5% Triton-X100. The GST- and HIS-fusion proteins on beads were stored at -20°C after addition of glycerol to 5%.Bacteria were transformed by electroporation and the colonies selected on LB-agar plates with ampicillin and kanamycin, each at 100 µg/ml, and chloramphenicol at 34600 = 0.8) in 2% galactose medium for the GAL-regulated constructs, or in YePD. The cells were harvested by centrifugation at room temperature for 5 minutes at 4,000 rpm, washed in 50 ml TE and resuspended in 600 µl extract buffer. 0.5–0.6 mg of glass beads were added and the cells were broken by vigorous vortexing for 25 minutes at 4°C. The extract was clarified by centrifugation at 12,000 g for 20 minutes at 4°C and protein concentration was measured with the Bio-Rad protein reagent. 5–10 mg of protein extract were taken for each GST pull-down in a total volume of 350–400 µl extract buffer. 30–50 µl of 50% Glutathione Sepharose 4B beads coupled to GST fusion protein were added to each sample and incubated at 4°C for 1–2 hours with very mild shaking. The samples were washed 6 times with extract buffer with 2.5% Triton X100. The pellet was resuspended in 30–50 µl sample buffer x2, boiled for 5 minutes and centrifuged for 3 minutes at high speed to remove insoluble material. The supernatant was separated on a 12% polyacrylamide SDS gel with protein size standards followed by WB analysis.Yeast cells were grown overnight to late log phase Click here for additional data file.Figure S2Ufo1-Ho-19S RP complex with yeast extract from Formation of SCFRPN1-GFP cells.GSTUfo1 or control GST beads were incubated with yeast extract from cells with tagged genomic RPN1-GFP that were cotransformed with pGFP-HO and with pMYC-CDC53. The bead fraction was analysed by Western blotting with anti-GFP antibodies to detect Rpn1 and Ho, with anti-myc antibodies to detect Cdc53, and with anti-Ddi1 antibodies.(TIF)Click here for additional data file.Figure S3GSTUfo1 bead fraction.Rpn12 is present in the  A further experiment in which GSTUfo1 and GST beads were incubated with yeast extract in the presence of GFPHo as in GSTRpn12. The presence of Rpn12 is an indication that the 19S RP is intact.(TIF)Click here for additional data file.


EBNA1 mRNA, thereby suppressing the production of antigenic peptides that the human immune system recognises as foreign. Here, Blondel et al. establish a yeast-based assay that recapitulates GAr-mediated suppression of EBNA1 mRNA translation and use it to identify doxorubicin and its active analogues as compounds that specifically interfere with GAr-mediated suppression of translation. Importantly, in mammalian cells, doxorubicin and its analogues stimulate EBNA1 expression in a GAr-dependent manner and overcome the GAr-dependent restriction of antigen presentation. Together, these results validate the yeast-based assay as an effective cell-based screening approach for compounds that interfere with EBV immune evasion and that might, therefore, provide treatments for EBV-related diseases, including cancer. Page 435Latent infection with Epstein-Barr virus (EBV) is associated with several human cancers, including nasopharyngeal cancer. During latent EBV infections, the glycine-alanine repeat (GAr) domain of the EBV nuclear antigen-1 (EBNA1) inhibits translation of


COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines. To establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM) resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC50 and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p<0.05 for both). Higher maintenance BLM concentrations were associated with higher IC50 and increased doubling times (p<0.05). Significantly reduced DNA damage (COMET and γ-H2AX assays), G2/M arrest, and apoptosis (p<0.05 for each set of comparison) following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05). Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in ICBleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis. Streptomyces verticillis . The slight increase in G2/M arrest at baseline (prior to high dose BLM treatment) for these BLM resistant sub-clones could be explained by chronic exposure to BLM. The fact that BLM-resistant cells were able to proliferate at a BLM concentration that was not viable for their parental lines suggests that evasion of cell cycle blockage could be another mechanism of resistance. Taken together, our results suggest that BLM resistant cells may have acquired enhanced ability to prevent/reduce DNA damage caused by BLM. This in turn, may have led to the reduced G2/M arrest and apoptosis. 50 values and doubling times observed in ACHN0, AHCN0.1 and AHCN0.25 cells. However, until these findings are confirmed in other sets of sub-clones, this will remain an interesting finding in need of validation. A stepwise dose escalating method for BLM resistance development may result in a dose-response relationship among BLM maintenance concentrations, IC50 values and doubling time in three of seven cell lines. The absence of reversibility in the other four cell lines could be the result of individual cell line differences, or perhaps these cell lines needed longer breaks from BLM exposure to develop BLM sensitivity again. To our knowledge, the literature on reversing BLM-resistance in cancer cell lines has been extremely limited. This observation speaks to the potential importance of continued BLM exposure when developing resistant sub-clones. Moreover, the result suggests that reversible mechanisms such as epigenetic rather than (permanent) genetic changes may be playing a role in maintaining some of the BLM-resistance. After removing maintenance BLM concentrations from all seven resistant sub-clones, there was partial reversal of ICin vitro model may not explain resistance in patients, but remains a critical first step in studying BLM resistance mechanism. Secondly, the amount of BLM treatment for each cell line corresponded to ten times the maintenance concentration for each BLM resistant sub-clone. There is the possibility that the treatment is not “acute” or high enough to elicit significant cellular response following the treatment. However, significant responses were seen in parental cells. Thirdly, individual cell line differences were not well-accounted, given the range of cell line origins. Since each cell line may have unique mechanisms that contribute to BLM resistance, this may explain some of the variations observed across experiments in this study. Fourthly, the cells used were not monoclonal. This may result in cells behaving differently upon BLM treatment. Yet, given that it was technically difficult to repopulate cells from a single cell that survived the dose escalation, we adopted this conventional approach of resistance development. This study has limitations. Firstly, an In summary, this study described some of the relationships between BLM resistance, BLM-induced DNA damage, cell growth rate, cell cycle distribution, and apoptosis. The reduced DNA damage, reduced G2/M arrest, and reduced apoptosis observed in BLM-resistant sub-clones following high dose BLM exposure suggest that acquired BLM resistance involves effective DNA damage reduction and G2/M cell cycle evasion. The seemingly reversible resistance observed in at least some of the BLM resistant sub-clones suggests that some of the BLM- resistance in our cell lines models may have utilized non-permanent mechanisms such as epigenetic changes to cope with chronic BLM exposure. Our results provide the foundation for future research in biomarkers of BLM resistance, which may ultimately lead to an improved rationale for personalized chemotherapy selection.


Retinal laser injuries are often associated with aberrant migration of the retinal pigment epithelium (RPE), which can cause expansion of the scar beyond the confines of the original laser burn. In this study, we devised a novel method of laser-induced injury to the RPE layer in mouse models and began to dissect the mechanisms associated with pathogenesis and progression of laser-induced RPE injury. We have hypothesized that the proto-oncogene receptor, c-Met, is intimately involved with migration of RPE cells, and may be an early responder to injury. Using transgenic mouse models, we show that constitutive activation of c-Met induces more robust RPE migration into the outer retina of laser-injured eyes, while abrogation of the receptor using a cre-lox method reduces these responses. We also demonstrate that retinal laser injury increases expression of both HGF and c-Met, and activation of c-Met after injury is correlated with RPE cell migration. RPE migration may be responsible for clinically significant anatomic changes observed after laser injury. Abrogation of c-Met activity may be a therapeutic target to minimize retinal damage from aberrant RPE cell migration. Lasers have been broadly applied in our world and laser instruments are being increasingly employed in a vast variety of fields, including military, health, educational, and commercial laboratories Exposure to laser can cause severe clinical ocular injuries that mostly damage the RPE layer by photothermal and photodisruptive mechanisms Hepatocyte growth factor (HGF), also known as scatter factor, originally discovered and cloned as a potent mitogen for mature hepatocytes In the retina, c-Met is mainly expressed in RPE cells In this study, we devised a novel method of laser-induced injury to the RPE layer in mouse models and began to dissect the role of c-Met in the pathogenesis and progression of late stage complication of laser-induced RPE injury. We have hypothesized that c-Met is intimately involved in the migration of RPE cells as an early response to injury. We demonstrate that retinal laser injury increases expression of both HGF and c-Met, and induces phosphorylation of the c-Met suggesting activity. Using transgenic mouse models, we show that constitutive activation of c-Met induces more robust RPE migration while abrogation of the receptor reduces these responses, suggesting that modulation of the c-Met activity may influence post-injury responses to laser burns. This study identifies c-Met as a potential therapeutic target to control aberrant RPE migration and wound enlargement after laser-induced injury.This study was approved by the IACUC of the Schepens Eye Research Institute and in accordance to the Declaration of Helsinki for the use of animals. All experiments were performed in accordance with the association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. In this study we compared three different types of mice which are detailed in fl/fl mice, a conditional knockout of c-Met, was obtained from National Cancer Institute . These mice were also backcrossed to B6 mice ×6 to produce a stable colony in the B6 background (c-Metfl/fl mice). In c-Metfl/fl mice, exon 16 of the c-Met genome is flanked by lox p sites. In presence of Cre recombinase , floxed p sites are permanently spliced out rendering c-Met inactive  with a wavelength of 810 nm, spot size of 350 µm, 150 mW power for 150 ms. 12 laser spots were applied in each animal, 3 per each retinal quadrant centered around the optic nerve . For shaAll mice were scarified by carbon dioxide inhalation at the different intervals from 0.5 hr to 14 days after the laser treatment. Eyes were quickly enucleated and fixed in 4% paraformaldehyde for histological examination. Some fresh retinas were collected to extract the mRNA for c-Met and HGF expression.in situ Cell Death Detection Kit .After fixing overnight in 4% paraformaldehyde, eyeballs were transferred into PBS buffer with 30% sucrose for one hour and processed for either paraffin or frozen sections at a thickness of 8 µm. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), a common method for detecting DNA fragmentation that results from apoptotic signaling cascades, was used to confirm areas of laser damage. To this end, cryosections of the retina of B6 mice were stained with Paraffin sections for IHC staining were warmed overnight, depraffinized with xylene, taken through serial alcohol dilutions and hydrated to distilled water. Sections were bleached for melanin pigment using an established protocol To determine whether laser injury affected expression of c-Met and HGF in the RPE monolayer and outer retina, laser-induced retinas of B6 mice were collected at the following time points, 0, 0.5, 1, 3, 6, 12 hr, 1 day and 14 days . Total mRNA was extracted using RNA 4 Aqueous kit . Reverse transcriptase reaction was performed for each mRNA sample using Retroscript kit . 1 µg of total mRNA was used as template to synthesize first-strand complementary DNA (cDNA). RT-qPCR was performed with 3 independent repetitions using the API Prism 7900HT Sequence Detection system  according to the instruction of SYBER Green PCR Master Mix . The reaction program included 2 min at 50°C, 10 min at 95°C, 40 cycles for 15 s at 95°C and 60 s at 60°C. The parameter threshold cycle was designed as the fractional cycle number, at which the fluorescence signals were generated during each PCR cycle. c-Met and HGF expression was calculated from the standard curve; quantitative normalization in each sample was performed using the expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control using the delta-delta method 10 Tu/ml for both AAV-Cre and AAV-GFP. Homozygous c-Metfl/fl mice were injected either with a mixture of AAV-Cre/AAV-GFP (ratio 9∶1) or AAV-GFP using a trans-scleral approach into the subretinal space under direct observation. The small amount of AAV-GFP in the AAV-Cre/AAV-GFP mixture induced a low-level background green fluorescence that can be detective in vivo by epifluorescence microscopy.AAV-Cre and AAV-GFP (serotype 2) were constructed and supplied by the Harvard Gene Intiative Core . AAV vectors were previously purified and titrated to about 1×10To perform this injection, a silk suture was penetrated through the upper eyelid. The eyelid was gently retracted and the eyeball was projected out from the eye socket to improve exposure. A small O-ring was placed on the eye. The pupil was dilated with one drop of each 2.5% phenylephrine and 0.5% tropicamide. Gonak  was applied over the O-ring to make an optical connection for visualization of the fundus. A sclerotomy (puncture hole) was made in the posterior portion on the wall of the eye using a 31G needle. A micro-glass pipette was used to deliver 2 µl of AAV solution through the sclerotomy into the subretinal space. Mice receiving subretinal injection of AAV-GFP served as controls for mice receiving AAV-Cre.fl/fl mice were subjected to laser treatment. The injected mice  were scarified on day 14 after laser treatment and examined expression of c-Met, p-Met and RPE65 in the outer retina and RPE layer.Analgesic buprenex (2 mg/kg) was administererd subcutaneously to mice before procedure and every 12 hr for 2 days. Antibiotic ophthalmic ointment  was applied to the eyes three times daily for 2–3 days postoperatively. Two weeks after subretinal injection, c-Met2) of c-Met, p-Met, HGF and RPE65 in the IHC stained slices were measured with ImageJ 1.46d (resolution at 300 pixels/mm). These included comparison of quantified measurements. Migrated cells in ONL and up to INL on the IHC stained sections were also manually counted using the image processing and analysis program ImageJ . One-Way ANAVO, Mann–Whitney U test and independent samples t-test were used to compare differences in the expression of aforementioned markers and the number of migrated cells. To establish linear regressions, log10-transformation was performed to normalize data and applied to the expression of c-Met, the days after the laser treatment and the number of migrated cells in B6 and TPR-Met mice. The data were presented as mean ± SD. All specific analyses and regressions were performed in SigmaPlot 11.0. The level of significance was set at 0.05.The respective expression areas   (There were no detectable changes in respective expressions of c-Met and HGF mRNA in B6 mice in the first 3 hr after the laser injury . The mRN U test) . At 3 hr U test) .2) and was significantly higher than the expression of c-Met or p-Met  at 3 hr after the laser injury. c-Met protein expression responded much slower and peaked at one day after injury (0.97±0.35 mm2). Expression of p-Met was constant within the first 24 hr (range: 0.28–0.51 mm2) after laser injury   .2) was detected in the control retina (2) (2) . The increase of c-Met expression  . After l (2) (2) . On day pression  was coinpression .2 vs. 0.62±0.05 mm2, independent samples t-test, P<0.05, 2 and 0.83±0.13 mm2, t-test, both P<0.05, t-test, P<0.05, Figure 5SS1). Prior to laser injury, there was low but detectable p-Met expression in untreated retinas of TPR-Met mice  was quite similar to control levels (0.39±0.08 mm2)  (2) compared to control  received only AAV-GFP injection without laser burns  and p-Met expression was very low (0.13±0.18 mm2). A second group of c-Metfl/fl mice (n = 5) received AAV-Cre/AAV-GFP (9∶1) injection without laser burns. In these mice no c-Met or p-Met was detected but RPE65 was expressed normally  . 14 days after the laser injury c-Met and p-Met expression of AAV-GFP injected mice increased comparing their control counterparts   followed by laser injury (2 weeks later). In these mice the expression of c-Met was very limited (0.13±0.08 mm2) (2)  increase in c-Met expression compared to controls  without any detectable p-Met expression  or changes in RPE65 expression .In this study, the mixture of 9∶1 AAV-Cre/AAV-GFP or AAV-GFP was subretinaly injected into homozygous c-Meter burns . In thesctively) . A thirdctively) . Howeverectively . A fourt.08 mm2)  and no p.08 mm2) . RPE65 emm2) (2) . OverallCryosections of B6 mouse retina were used to count the number of migrated cells in response to the laser injury. On day 7 after injury, migrated cells were observed in the ONL. IHC confirmed that these cells were indeed RPE. Furthermore, it indicated that c-Met green, , p-Met  and RPE6t-test, both P<0.05, at left side of t-test, P>0.05, fl/fl mice, few c-Met positive cells (4±1 cells) were found in ONL of AAV-Cre injected mice  , and more cells were identified on days 7 (6±1 cells) and 14 (8±1 cells)  . No p-Me = 0.03) .10-transformation was applied to both the days and c-Met expression from day 3 to day 14. Linear regression analysis confirmed that c-Met expression was significantly negatively related with the time (days) after the laser treatment in B6  and TPR-Met mice   . Further = 0.52) . This ob2 = 0.62, insert graph in fl/fl mice tapered RPE migration after laser injury.Interestingly, a significant linear relationship was confirmed between the expression of c-Met and the migration of RPE cells in TPR-Met mice (y = 0.83× +1.02, RLasers have been broadly applied in our world and laser instruments are being increasingly employed in a vast variety of fields, such as military, health, educational, and commercial laboratories Laser-induced retinal alternations characterized to date indicate that with increased energy, damaged areas extend to outer segment layers of the retina in addition to the RPE, which itself is considered the primary site of absorption RPE cell migration and proliferation are believed to play a role in expansion of laser scars and pathogenesis of PVR Human vitreous contains not only mitogens for RPE cells but also factors that mediate their migration. Clinically, the appearance of RPE cells in the vitreous may be a consequence of injury or rhegmatogenous retinal detachment in which these cells now become exposed to the vitreous fl/fl floxed mice reduced RPE migration into the wounded sites.In vitro studies have shown that RPE cells can transdifferentiate to either neurons or lens cells in culture c-Met participates in cell growth and migration during embryonic development, and plays a significant role in skin regeneration process. The c-Met protein also known as HGF receptor encodes for a thyrosine kinase receptor which is activated by HGF. Birchmeier et al. showed that receptor-type tyrosine kinases are critical in regulating epithelial differentiation and morphogenesis HGF is the only known ligand for the c-Met receptor. c-Met is normally expressed by cells of epithelial origin There are situations in vivo in which RPE cells may migrate- development Clinically, RPE cells can migrate anywhere in the retina. RPE cell migration may be mediated through the activation of the c-Met receptor. In this scenario, c-Met activation induces transdifferentiation of RPE cells and its migration across the all retinal surfaces. In response to retinal laser injury, c-Met receptor system is activated through release of HGF and is intimately involved in the responses of RPE to laser injury. This is supported by the observation that constitutive activation of c-Met increased RPE migration into the retina and abrogation of the receptor diminished RPE cell migration. Therefore, control of c-Met activity may be a future therapeutic target to minimize retinal damage that may ensue after laser injury.


Heroin addiction is a chronic, complex disease, often accompanied by other concomitant disorders, which may encumber effective prevention and treatment. To explore the differences in expression profiles of serum proteins in control and heroin addicts, we used two-dimensional electrophoresis coupled to MALDI-TOF/TOF, and identified 4 proteins of interest. Following validation of the increase in serum transthyretin, we assessed serum levels of thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4), and observed a robust increase in T4 in heroin addicts compared to controls. In addition, we performed haptoglobin (Hp) phenotyping, and showed that the frequency of Hp0 (serum devoid of haptoglobin) was significantly higher in heroin addicts. Altogether, these findings indicated that: (1) thyroid hormone imbalance is present in heroin addicts; (2) anhaptoglobinemia (Hp0) might a risk factor or a deleterious effect of heroin abuse. World Drug Report 2013 (United Nations Office on Drugs and Crime). Even worse, the number of registered heroin consumers keeps rising rapidly, reaching 1.272 million by the end of 2012. As an illegal and highly addictive drug, heroin causes serious medical, economic and social problems, threatening the society Heroin is the most prevalent drug of abuse in China, according to Interestingly, genetic predisposition is a major contributing factor of the development of addiction. Epidemiological studies in men demonstrated that approximately 40–60% of developing an addiction to heroin is genetically determined The deleterious consequences of heroin dependence are largely known as cognitive deficits and lethality to the individual, and impairment of economy and public safety to the society. Studies have shown that parts of the central nervous system, including dynorphin-kappa opioid system The emergence of the terms “morphinome” and “cocainomics” underscored the importance of proteomic research in opiate addiction To uncover proteins potentially involved in heroin addiction, we utilized a two-dimensional electrophoresis (2-DE)-based proteomics approach to examine differential protein expression in the serum of heroin addicts and healthy controls. Following the identification differentially expressed proteins, we validated the increase in serum transthyretin levels in heroin-addicted patients, and further found that thyroid hormone thyroxine (T4) was elevated. In addition, we performed haptoglobin phenotyping, and showed, for the first time, that patient serum devoid of haptoglobin (anhaptoglobinemia) positively correlated with heroin dependence. These findings indicate that thyroid hormone balance is disrupted in heroin addicts, and that anhaptoglobinemia might be a genetic risk factor or a pathological consequence of the disease, which could provide clues for the prevention or treatment of heroin addiction.This study was approved by the Human Subjects Division at Peking University Health Science Center. All subjects were of Han Chinese descent. To preclude the effect of gender on the serum proteome, only male individuals were included in this study g (4°C) for 10 min. Serum used for proteomic experiments were additionally re-centrifuged at 1,300×g (4°C) for 10 min to remove all residual cell debris. The resulting supernatants were aliquoted and frozen at −80°C until use.Approximately 5 ml of blood was drawn by venipuncture, and collected into unheparinized tubes. According to the Human Proteome Organization (HUPO) reference sample collection protocol Twenty samples were randomly selected from each group for 2-DE. Each sample corresponded to one gel. Protein extracts from control and patient serum were processed in parallel to ensure maximal comparability. Albumin was removed from serum samples as described previously P<0.05).Protein spots of interest were excised from 2-DE gels, washed, dried, and enzymatically digested with trypsin. The extracted peptide solution was subjected to the Applied Biosystems 4700 Proteomics Analyzer MALDI-TOF/TOF for MS/MS analysis. The peptide masses were searched against the National Center for Biotechnology Information (NBCI) database using Mascot Search from Matrix Science. A criterion for significance was defined using a probability-based molecular weight search (MOWSE) score. Individual scores greater than 56 indicated identity or extensive homology  membranes . TTR  was detected using a polyclonal rabbit anti-prealbumin antibody (Santa Cruz), followed by incubation with a secondary HRP-conjugated goat anti-rabbit antibody (Proteintech Group). To ensure equal protein loading, membranes were also probed with a polyclonal rabbit anti-GAPDH antibody  For each participant, serum levels of thyroid stimulating hormone (TSH), triiodothyronine (T3) and thyroxine (T4) were determined by radioimmunoassay. The assay was performed according to manufacturer’s protocol , with intra-assay coefficients of variation less than 10.0% and inter-assay coefficients of variation less than 15.0%.g (4°C) for 40 min, and the resulting supernatant was aliquoted and stored at −80°C until use. The Hp-Hb complex solution was prepared by adding 2 µl of 10% Hb solution to 10 µl of serum, and mixing for 5 min at room temperature 3[Fe(CN)6], and 300 µl of 30% H2O2.All serum samples from each group were subjected to haptoglobin (Hp) phenotyping. A 10% hemoglobin (Hb) solution was prepared as previously reported t-test was performed. In Hp phenotyping, the chi-square (χ2) goodness-of-fit test (one sample test) was used for each single Hp phenotype distribution, and the Fischer’s exact test was used to compare Hp phenotype frequencies between the two groups. A P value of less than 0.05 was considered statistically significant.Statistical analysis was performed with the SPSS Statistics software (version 17.0). Unless otherwise indicated, the two-tailed Student’s P<0.05 was considered significantly differentially expressed between heroin addicts and healthy controls, which included spot numbers 2, 3, 4, 7 and 8 .The eight spots were excised from the gels, digested with trypsin and analyzed with MALDI-MS. Peptide masses were used to query the NCBI database, and the eight spots were all significant with MOWSE scores greater than 56. The results, including protein names, accession numbers, the number of matching peptides, the percentage of sequence coverage, the theoretical MW and pP<0.01), which is consistent with the results from 2-DE  is one of the three thyroxine (T4)-binding proteins in human serum 2 chain of the protein, whereas spot 8 is the α1 chain As shown in The three major Hp phenotypes  and serum not expressing Hp (Hp0) were easily distinguished by characteristic patterns of bands representing the Hp-Hb complexes . The freP value of 0.0514, which is close to the critical value of 0.05, suggests that there might be potential differences in the distribution of Hp phenotypes if further increasing sample size. These results indicate a possible correlation between the lack of Hp and heroin addiction.Next, we assessed the distribution of each Hp phenotype using the chi-square goodness-of-fit test (one sample test). Hp1-1, Hp2-1, Hp2-2 frequencies did not show marked differences between the two groups, whereas was found to be significantly differently distributed (P≈0.025). Fisher’s exact test was used to compare the distribution of Hp phenotypes in cases and controls. This test was preferred over a parametric test because there were instances of small cell counts in the tables. No substantial differences were revealed. However, a Heroin addiction is a major public health problem worldwide. It is widely believed that the mesolimbic dopamine pathway plays a pivotal role in drug reward and addiction, and that dopamine release is central to the reinforcing effects of drugs of abuse Proteomic techniques are broadly applied in human disease for the discovery of differentially expressed proteins. Here, we used 2-DE and tandem MS, and identified 4 proteins (peptides) of interest. They were immunoglobulin J chain, transthyretin, haptoglobin, and vitronectin. As opposed to gel-free proteomics, this detection method sets the detection threshold at a very high level, which can be easily converted and applied in a clinical setting.Transthyretin (TTR) is a 55-kDa homotetramer with a dimer of dimers configuration, and sometimes exists in the form of monomers as well 1, 8.9 kDa; α2, 16 kDa) and β (40 kDa). The β chains are identical in all Hp phenotypes, and variations are solely caused by the presence of different α chains. Three major Hp phenotypes are known to exist: Hp1-1, Hp2-1 and Hp2-2. Some individuals do not express Hp (anhaptoglobinemia), which is denoted by Hp0. In this study, we demonstrated that the frequency of Hp0 phenotype was significantly higher in heroin addicts than in healthy controls, which is the first report on anhaptoglobinemia in the context of heroin addiction. However, whether it is a risk factor conferring vulnerability to addiction, or a consequence of heroin use Haptoglobin (Hp) is an acute phase protein that binds free hemoglobin and eliminates it from the circulation to prevent kidney injury and iron loss The other two proteins (peptides) identified were immunoglobulin J (IGJ) chain and vitronectin. IGJ participates in the polymerization of the antibodies Immunoglobulin A (IgA) and Immunoglobulin M (IgM). Serum IgA is a monomer, and does not require the presence of IGJ chains. Therefore, the identified IGJ chains in our study originated from IgM. Wetli et al. conducted immunological studies in 40 heroin addicts. Serum IgM and IgG increased by 80% and 37.5%, respectively In summary, the 2-DE/tandem MS approach applied in the present study provided a useful tool to identify proteins related to a particular disease state, and to provide clues for in-depth analyses of proteins of interest. By use of this strategy, we were able to show, for the first time, that T4 and its carrier protein, TTR, were elevated in the serum of heroin addicts, and that the occurrence of Hp phenotype Hp0 (anhaptoglobinemia) is associated with heroin addiction. Additional studies need to be carried out to figure out how heroin dependence affects thyroid balance. Furthermore, the currently unclear causal relationship between anhaptoglobinemia and heroin addiction warrants deeper investigation.


AIDS-PCNSL is a rare EBV-associated B-cell neoplasm that continues to carry a poor prognosis, even in the highly active antiretroviral therapy (HAART) era. We hypothesized that overall survival (OS) is affected by prior diagnosis of central nervous system (CNS) infections as well as treatment approaches to both HIV and AIDS-PCNSL. We evaluated risk factors and temporal trends for OS in patients with AIDS-PCNSL.Adults with AIDS-PCNSL were identified through a computer linkage that matched AIDS case diagnosed between 1990-2000 from the San Francisco adult AIDS case registry with the California Cancer Registry (1985-2002), with mortality follow-up through 12/31/2007. Patients with non-B-cell histology or history of systemic non-Hodkgin lymphoma diagnosed within 2 years prior to AIDS-PCNSL diagnosis were excluded. Prognostic factors evaluated include: diagnosis of CNS infection prior to AIDS-PCNSL, diagnosis of other common opportunistic infection (OI) prior to AIDS-PCNSL (pneumocystis pneumonia [PCP] or mycobacterium avium complex [MAI]), pathologic versus clinical diagnosis, receipt of cancer therapy, HAART prescribed prior to or within 30 days of AIDS-PCNSL diagnosis, and year of diagnosis . Survival analyses employed Kaplan-Meier methodology.A total of 207 patients were identified, 96% male and 4% female. Median age 39 (IQR 35-46), 68% white, 21% black, 20% Hispanic, 2% Asian. Median CD4 20 cells/uL (IQR 6-53). HIV risk group: 79% MSM, 8% IDU, 9% MSM/IDU. CNS infections prior to AIDS-PCNSL: toxoplasmosis 8%, cryptococcus 9%, histoplasmosis 1%, extrapulmonary tuberculosis 1%. Treatment category: none 42%, radiation only 52%, chemotherapy 6% . Risk factors for OS included prior CNS infection (p<0.0001), HAART (p=0.0023), AIDS-PCNSL treatment (p<0.0001), and calendar period of AIDS-PCNSL diagnosis (0.001), but not prior PCP or MAC (p= 0.23). Figures . OS was Prior diagnosis of CNS infection, HAART, and cancer treatment are strong predictors of OS. OS improved over time in these patients. Earlier diagnosis of AIDS-PCNSL and/or CNS infection, treatment of CNS infections, and cancer treatment that includes HAART and concomitant chemotherapy may increase AIDS-PCNSL survival. Prospective evaluation of curative-intent chemotherapy-based approaches to AIDS-PCNSL is urgently needed. Additional analyses are ongoing.


The aim of this study is to evaluate in-house antibiotic use in a state hospital in Turkey with its cost, using the ATC/DDD index, which is an accepted standard method.This study was performed as a point prevalence study in a state hospital with 372 beds. All in-house patients using antibiotics on July 19, 2011 were included in the study. Indications for antibiotic use and information about the patients were recorded on special forms. Antibiotic use and cost analysis were evaluated using the ATC/DDD index, which is also suggested by the WHO to be used in similar studies.147 patients out of 308 patients who were in-house were identified to use antibiotics with appropriate indications for prophylaxis or treatment in 61% of the patients. The rate of appropriate antibiotic use was identified to be in 78%, while this rate was 38.9% in surgical clinics. The daily cost of the antibiotics consumed on the date of the study was calculated as 4104.79 TL (=2476.80 USD).The rate of inappropriate use of antibiotics seems to be high in our hospital. This will result in both increased costs and also increased nosocomial infection rates with resistant species. Infectious disease specialists should take more active roles in the in-house antibiotic use, hospitals should prepare and implement their own principles of antibiotic use, and microbiology laboratories should be used more effectively. These measures would decrease the conspicuous shortcomings in the antibiotic use. As the anti infective agents are most frequently used group of drugs in our country, also doctors most frequently error when chosing them ,2. ImproUnnecessary use of antibiotics is a major worldwide problem. Despite of detailed research on trends of antibiotic use in and out of the hospitals in many countries, no sufficient data is available for our country .Because each antibiotic has different unit dose of daily administration, a specific standardized method should be used in the evaluation of in-hospital antibiotic use. Of the current standard methods, the method suggested by the World Health Organization (WHO) is the prominent. The ATC/DDD index is developed and intervaly updated by the WHO. The WHO Collaborating Centre for Drugs Statistics Methodology has standardized this with the ATC/DDD  index . DDD 100The aim of this study was to evaluate the use and cost of antibiotics in a Turkish state hospital by a point prevalence research with the ATC/DDD index which is accepted as a standard.Isparta State Hospital is a secondary healthcare facility with 372 bed capacity and 4 different intensive care units with 21 bed capacity.A research of modified point prevalence research was carried out in order to evaluate the frequency and appropriate use of antibiotics in our hospital. The data of this study were obtained from 7 departments of internal diseases , infectious diseases), 8 departments of surgical diseases , cardiovasculer surgery(CVS), thoracic surgery, general surgery, orthopedics, urology and plastic surgery) and 4 intensive care units . Data concerning antibiotic use in the hospital were collected on the 19th of July 2011 by an infectious diseases specialist and all patients receiving antibiotics were included to the study. Data were collected from patient charts using a standard form; patients personal information, underlying disease, the name of the antibiotic in use, dosage, usage, duration, reason of antibiotic use  and microbiological test results were recorded on the form.Th existence of an infectious disease was detected by evaluating patient’s clinical complaints, physical examination findings and laboratory findings all together. In diagnosing nosocomial infections, CDC (The Centers for Disease Control) criteria and NNIS  methodology were used . The antSurgical prophylaxis was evaluated taking into account the drug dose, administration way, duration of prophylaxis and international guidelines .Patients using antibiotics were evaluated according to suitable indication, dose, administration way and criteria for adequate antibiotic. When evaluated by the infectious diseases specialist, patients matching all criteria were accepted as “suitable”, and in the absence of even one criteria the patient was “non-suitable”.The DDD is the assumed average maintenance dose per day for a drug used for its main indication in adults. Only drugs with an ATC code can have DDD values. The DDD value in grams of every drug is defined by the WHO and is periodically updated. DDD values of every antibiotic is calculated separately .Defined Daily Doses (DDDs) are calculated separately for every antibiotic, the average maintenance dose for an adult weighing 70 kg is prepared in main indications and the active substance should be taken as grams (or I.U.).DDDs = Number of boxes x number of tablets in the box or number of vials x tablets in grams or the weigh of the vial / the DDD value of the antibiotic in grams.In this calculation method, the form used for in-bed patients is the ratio of the total DDD per 100-bed-days. This index is called antimicrobial consumption index (ACI).Also, the antibiotic consumption index of a country or geographical area at a certain period of time is calculated by DDD per 1000 people.The number obtained is the antibiotic consumption index of that hospital/clinic or population.In this study, DDDs of anti infective agents are listed for systemic use according to ATC/DDD 2010 Index.The total cost of every antibiotic used in the hospital was calculated according to the price list of the General Directorate of Pharmaceuticals of The Republic of Turkey on the 19th of July 2011. Afterwards, the cost was converted to USD according to the exchange rates of the Central Bank of The Republic of Turkey on the 19th of July 2011 .The mean age of the 308 patients was 56.7 + 18.2. Of the patients included to the study, 161 were male (52.3%), and 147 were female (47.7%). Of the 308 patients, 147 (47%) received antibiotics for any reason. Of the 147 patients, 47 (32%) received antibiotics for surgical antimicrobial prophylaxis, 92 (62.6%) empirical antimicrobial therapy and only 8 patients (5.4%) received antibiotics based on microbiological data.When evaluating the 147 patients using antibiotics, 61.9% were evaluated as “appropriate” and the rate in the clinics of internal diseases was 76.6%, in the surgical clinics 38.9%, in the intensive care units 81.8%. According to the microbiological data, of the 8 patients receiving antibiotics 5 are intensive care patients, and while 45.5% of the patients from the intensive care units receive antibiotics according to microbiological data, in the departments of internal and surgical diseases this is only 2.6% and 1.7% respectively . Duration of surgical prophylaxis according to the clinics was; orthopedics 9.5, thoracic surgery 6.8, general surgery 2.1, urology 1.4, ENT 1.3 days, respectively. The only service where third generation cephalosporins were used in surgical prophylaxis was thoracic surgery.In our study, cephalosporins were found to be the most frequently used antimicrobials among all antibiotics, with a rate of 57%. Among the cephalosporins, 3rd generation cephalosporins (including beta lactamase inhibitors) constituted 21% of all antibiotics used. First generation cephalosporins were most frequently used with a rate of 20.8%. Fluoroquinolones and penicillins (including beta lactamase inhibitors) were the groups prefered after cephalosporins. In the departments for internal diseases most frequently preferd antibiotics were 2nd generation cephalosporins and fluoroquinolones, in the surgical departments 1st generation cephalosporins, and in the intensive care units 3rd generation cephalosporins (including beta lactamase inhibitors) and carbapenems. The cost per infected patient was 27.92 TL (=16.89 USD). In the intensive care unit daily antibiotic costs were 546.08 TL (=329 USD), per infected paient daily costs were 49.64 TL (=29.95 USD). In the departments of internal diseases daily antibiotic costs were 2793.08 TL (1685 USD), per infected patient being 36.27 TL (=21.89 USD). Among the departments of internal diseases, the department for chest diseases had the most frequent antibiotic use and te highest number of in-bed patients. The daily antibiotic costs of the department for chest diseases was 1126.45 TL (679.69 USD), of which 20.48 TL (=12.36 USD) per patient/day. Total antibiotic costs per day in the surgical departments were found to be 765.63 TL (= 463.98 USD), of which per infected patient 12.98 TL (=7.86 USD) and penumonia (14.3%) draws our attention. High rate of antibiotic use is acceptable because one of the major pharmacologic treatment used in acute exacerbation of chronic obstructive pulmonary disease(COPD) are antibiotics .When we evaluated the duration of surgical prophylaxis as a whole, the proper antibiotic choice, administration time and way were found to be only 27.7% appropriate. The major reason of the low proper antibiotic use rate in the surgical departments compared to other departments were the errors in the use of surgical prophylaxis. While the antibiotic for surgical prophylaxis was largely properly chosen, what was improper was the duration of prophylaxis. The mean duration of surgical prophylaxis was found to be 4.74 days. In surgical units, where surgical prostheses are implanted, surgical prophylaxis has been observed to be more inappropriate. Surgical prophylaxis was most frequently used in the orthopedics service. Average duration of surgical prophylaxis was 9.5 days and all of the patients were using more than one drug combination containing antibiotics. Urology and ENT were services that implement the most appropriate surgical prophylaxis. In similar studies, no errors were seen in the agent used in the prophylaxis, while the unnecessary extension of therapy was the main problem ,29. The According to our study cephalosporins constitute 57% of the antimicrobials used in our hospital. Cephalosporins are followed by fluoroquinolones with 14.7% and penicillins with 10% of use. Of the cephalosporins used, 21% were 3rd generation, 20.8% 1st generation and 15.2% 2nd generation, which ranks on the first 3 places of the total drugs used. According to the ARPAC  project, all hospitals most frequently used penicillins, followed by non-penicillin beta lactams and fluoroquinolones . AntibioThe ACI in our hospital was found to be 55.1 DDD/100 bed-days. In the intensive care units the ACI was 87.8 DDD/100 bed-days, in the departments for internal diseases 56.4 and in the surgical departments 49.1 DDD/100 bed-days. Eventhough the proper antibiotic use in the surgical departments was found to be lower than the department for internal diseases and intensive care units, the ACI value was lower. According to Akalın et al.’s study  in a uniIf we keep in mind that antibiotics are the most frequently used drugs in the hospitals, than we would know that they constitute an important part of the total drug expenditure. In our hospital the defined cost per infected patient per day was 16.85 USD. As expected, the antibiotic use in the intensive care units for infected patients was higher than in other clinics. Antibiotic use in the surgical departments was found to be 7.86 USD per infected patient per day, which is lower than other departments. Despite of the unnecessary extension of surgical prophylaxis, the reason of this is the choice of less expensive 1st generation cephalosporins. However, as the improper use of antibiotics is still high, the in-hospital antibiotic expenditure can be reduced even more. Reducing improper use of antibiotics would prevent the incidence of life-threatening serious infections, also preventing long hospital stays and higher health expenditures. The department for pulmonary diseases draws attention with the highest number of patients and also with the highest number of patients using antibiotics. The department for pulmonary diseases uses 27.5% of the daily antibiotic costs. Vast majority of the patients are hospitalized in this department due to diseases caused by smoking. Smoking can be considered as one of the factors that increases the use of antibiotics in hospitals.Our study, carried out with point-prevalence method has certain limitations such as being single-centered, not having resistance ratios calculated and involving only one day of the year. The study was carried out in the summer period. Especially pneumonia and acute exacerbation of COPD are diseases seen more oftenly in the winter, and clinically more complicated diseases are less encountered in the summer period. However, by applying a method inexpensive and easy to implement as the point-prevalence, we can say that we gained detailed knowledge of the antimicrobial use in our hospital.As a result, the use of ATC/DDD system in hospitals would provide internationally valid data in the evaluation of antimicrobial use. We believe that, more efficient utilization of infectious diseases experts in the use of antibiotics in hospitals, creating of guides for antibiotic use specific to every hospital and more efficient use of the microbiological laboratories may be of benefit in the resolving of existing problems.Written informed consent was obtained from the patient’s for the publication of this report.The authors have no competing interests to declare.HS involved in designing of the project, collection of data, data analysis and interpretation, and write up of the manuscript. IG designed the study, and prepared the manuscript for publication. Other Authors: Collection of data. All authors read and approved the final manuscript.


In conclusion, increased IgG4+ cells are not uncommonly seen in sinonasal or orbital/periorbital biopsies of GPA, which could pose as a diagnostic pitfall.Granulomatosis with polyangiitis (Wegener's) (GPA) may mimic IgG4-related disease (IgG4-RD) on histologic examination of some biopsies, especially those from head and neck sites. IgG4 immunostain is often performed in this context for differential diagnosis with IgG4-RD. However, the prevalence of IgG4+ cells in GPA has not been explored. We examined the IgG4+ cells in 26 cases confirmed as GPA by a thorough clinical and pathologic assessment. Twenty-six biopsies consisted of 14 sinonasal/oral cavity/nasopharynx, 7 orbit/periorbital, 3 lung/pleura, 1 iliac fossa/kidney, and 1 dura specimens. Eight of 26 (31%) biopsies revealed increased IgG4+ cells (>30/HPF and >40% in IgG4+/IgG+ ratio). The IgG4+ cells and IgG4+/IgG+ ratio ranged 37–137/hpf and 44–83%, respectively. Eight biopsies with increased IgG4+ cells were from sinonasal ( Granulomatosis with polyangiitis (Wegener's) (GPA) is an immune-mediated systemic necrotizing vasculitis often affecting the upper respiratory tract, lung, and kidney . InvolveOn histologic examination, GPA can mimic IgG4-related disease (IgG4-RD) since the inflammatory background in GPA may be rich in plasma cells and accompanied by fibrosis and/or obliterated blood vessels as in IgG4-RD , 3, 4. Sn = 16), oral cavity (n = 1), orbital region (n = 11), lung/pleural (n = 6), kidney (n = 1), and dura (n = 1). Glass slides from each biopsy were reviewed and reassessed for the following histologic features: geographic necrosis, necrotizing granulomas, vasculitis, multinucleated giant cells, microabscesses, and fibrosis. Based on these histologic parameters, each case was scored as to the confidence of histopathologic diagnosis of GPA as follows: 0 = nondiagnostic biopsy or not GPA with some findings against the diagnosis of GPA such as infection, 1 = nonspecific (one feature present); 2 = suggestive of GPA (two features present), or 3 = consistent with GPA (three or more features present). A definitive clinicopathologic diagnosis of GPA was made if a case had met the modified clinical criteria of the American College of Rheumatology (ACR) for GPA [ Cases with the diagnosis of GPA were identified via electronic search from our surgical pathology file. We initially retrieved 36 biopsies from various body sites obtained during a period of 1999–2011. The sites of biopsies included sinus/nasopharynx . Monoclonal IgG4 antibody  and polyclonal rabbit antihuman IgG antibody  were used for the study. IgG4+ and IgG+ cells were counted using the following steps.Areas of highest density of IgG4+ plasma cells “hot-spot” were identified at low power magnification.2), three high-power fields (HPFs) of IgG4+ hot spots, and their corresponding areas of IgG immunostain were photographed.Using 40x objective lens  and immunoglobulin subclasses.n = 4) and orbital/periorbital (n = 4). The IgG4+ cell count ranged from 0 to 28/HPF in the remaining 18 biopsies that were from sinonasal/oral cavity/nasopharynx (n = 10), orbit/periorbital (n = 3), lung/pleura (n = 3), iliac fossa/kidney (n = 1), and dura (n = 1). The IgG4+ cell counts and IgG4/IgG ratios in the cases without increase in IgG4+ cells ranged from 0 to 28 per HPF and from 0 to 39%, respectively (Eight of 26 biopsies (31%) showed increased IgG4+ cells with an average IgG4+ cell count and an IgG4+/IgG+ cell ratio ranging from 37 to 137/HPF and from 44 to 83%, respectively . These 8ectively . There wectively ; 2 of 8 The 26 biopsies in this study were obtained from 23 patients. The status of serum CRP level was assessed at the time of the biopsy in 18 patients, and it was elevated in 15 of them (83%) . ImmunogIn this study, we sought to determine the prevalence of increased IgG4+ plasma cells in GPA in order to address the role of this finding in the differential diagnosis with IgG4-RD. We believe that this is the largest series of GPA cases to examine IgG4+ cells with application of current criteria and method for evaluation of increased IgG4+ cells in the setting of IgG4-RD. An increase in IgG4+ cells in GPA has been suggested in a recent study by Vaglio et al.  in whichIn our study, we applied the current criteria and methods in counting IgG4+ cells, which should provide useful and important information in routine diagnostic surgical pathology practice. Also, we made an extra effort to ensure the diagnosis of GPA by a thorough clinicopathologic evaluation and only included the cases with irrefutable diagnosis of GPA. All but one case showed positivity for C- or P-ANCA confirmed with ELISA for PR3 or MPO; the single ANCA negative case demonstrated definite histopathologic findings (pathologic diagnosis score 3) as well as clinical features for GPA, which supported our diagnosis of GPA. Moreover, all 8 cases with increased IgG4+ cells in this study were positive for ANCA, which reiterated our point.2 versus 0.088 mm2, resp.). Therefore, the threshold used in this study would be comparable to the one of the most stringent ones in the literature.The cutoff number of increased IgG4+ plasma cells has not been well established, and many studies have suggested different cutoff points. Kamisawa et al. and Zhang et al. have used a cutoff point of >30 per HPF , 11  serum IgG4 concentration of >135 mg/dL and (2) >10 IgG4+ plasma cells/HPF with a ratio of IgG4+/IgG+ cells >40% . IgG4 sePositive ANCA has been reported in patients with Grave's disease receiving antithyroidal medication such as propylthiouracil (PTU) , 17 and Previous studies have reported the presence of inflammatory infiltrates rich in IgG4+ plasma cells in clinical settings other than IgG4-RD –21, usuaIn vitro, ANCA activated neutrophils by colligating PR3 and FcγRIIa/IIIb receptors [in vitro test, or neutrophil FcγRI expression [γRIIa/IIIb or colligation of other unidentified cell surface molecules [The predominance of IgG4 as well as IgG1 subclasses of ANCA was first reported in patients with GPA and other clinically related disorders by Brouwer et al. in 1991 . Hollandeceptors . ANCA arpression . This stolecules . Liu et olecules . They re Given these serological and immunological findings, one can postulate a possible role of tissue infiltrating IgG4+ cells in the pathogenesis of GPA. However, the underlying cause or precise mechanism for increased IgG4+ cells in the tissue as seen in some of our GPA cases has not been completely elucidated and further study would be needed in the future. Also, whether there is any pathogenetic relationship between GPA and IgG4-RD is not entirely clear. Although there have been studies reporting elevated IgG4 in the setting of ANCA-associated systemic diseases, no study demonstrating elevated ANCA in IgG4-RD exists in the English literature to our knowledge. Our anecdotal experiences also indicate that the ANCA is generally not elevated in IgG4-RD.In conclusion, one should be aware of the fact that IgG4+ cells can be remarkably increased in biopsies of GPA of the sinonasal and orbital/periorbital regions. Since the morphologic and clinical manifestations of GPA and IgG4-RD may overlap, it could be a significant diagnostic pitfall in the differential diagnosis of these two entities. Further study is needed to confirm our observation in a larger number of cases, and a further exploration of potential pathogenetic relationship between GPA and IgG4-RD might be of interest as well.