Critical illness polyneuropathy (CIP) and critical illness myopathy (CIM) are complications causing weakness of respiratory and limb muscles in critically ill patients. As an important differential diagnosis of Guillain-Barré syndrome (GBS), CIP and CIM should be diagnosed with caution, after a complete clinical and laboratory examination. Although not uncommon in ICU, CIP and CIM as severe complications of percutaneous nephrostolithotomy (PNL) have not been documented in literature.A 48-year-old Chinese woman was referred to our hospital, complaining of occasional pain in the right lower back for one month. Lithiasis was diagnosed by ultrasonographical and radiological examinations on the urinary system. PNL was indicated and performed. The patient developed CIP and CIM on the fourth day after PNL. Early recognition and treatment of the severe complications contributed to a satisfactory recovery of the patient.This case expands our understanding of the complications of PNL and underscores the importance of differentiating CIP/CIM from GBS in case of such patients developing weakness after the treatment. Clinical characteristics and examination results should be carefully evaluated to make the diagnosis of CIP or CIM. Both anti-septic prophylaxis and control of hyperglycemia might be effective for the prevention of CIP or CIM; aggressive treatment on sepsis and multiple organ failure is considered to be the most effective measure to reduce the incidence of CIP/CIM. Critical illness polyneuropathy (CIP) and critical illness myopathy (CIM) are frequent complications of critical illness involving both motor and sensory axons,2. The cPercutaneous nephrostolithotomy (PNL) is a procedure requiring indications of the treatment of large hard infected stones, obstruction-related stones, extracorporeal lithotripsy failures and stones related with anatomic variations. Most ofA 48-year-old Chinese woman was referred to our hospital, complaining of occasional pain in the right lower back for one month. The pain was blunt, lasting several minutes each time. The episodic pain occurred 5–6 times altogether. The patient was otherwise healthy, without any remarkable history of diseases. Ultrasonography of the urinary system revealed enlarged right kidney with dilated pelvis and ureter, and multiple calculus in the right kidney and ureter. Intravenous urography (IVU) following a KUB showed hydronephrosis and a 2.5 cm × 1.0 cm high-density mass in the right ureter at the L3 level  was administered to control the infection. On the 2nd day after PNL, the patient’s conditions deteriorated even further; delirium, irritability and dyspnea appeared. The oxygen saturation decreased sharply to 60% and the patient fell comatose. Intubation and mechanical ventilation were immediately initiated. Five percent sodium bicarbonate (12.5 g) was intravenously administered to correct acidosis. The patient’s blood pressure could not be maintained above 90/60 mmHg by continuously infusing dopamine and metaraminol, use of methylprednisolone  was initiated. On the 3rd day after PNL, her blood WBC count was increased to 24.66 × 109/L  and platelet count decreased to 32 × 109/L. Level of creatinine in blood was elevated to 237.8 μmol/L. During the fourth day after PNL, absence of bilateral deep tendon reflexes was noted, whereupon neurologists were consulted. The Glasgow coma scale score was 7. She had even pupils with normal light reflex. Deep tendon reflexes were absent and pyramidal signs were negtive bilaterally; the Kennedy sign was positive. Since the patient was comatose, we could not examine the muscle strength of her extremities. ECG did not reveal any abnormalities. Laboratory tests showed an elevated serum level of aspartate transaminase (AST) , creatine kinase , CK-MB , and CK-MM . Although CIP/CIM were suspected, Guillain-Barré syndrome (GBS) could not be excluded. IgG antibodies against GM1, GM1b, GD1a, and GalNAc-GD1a in serum, were negative. Considering the patient’s critical situation, electrophysiological investigations and lumbar puncture were not performed though advised. Intravenous immunoglobulin (0.4 g/(kg body weight)/day, i.v.d., for 5 days) was empirically administered. On the 7th day after PNL, a methicillin resistant strain of S. aureus. was identified from her blood cultivation. Treatment combining vancomycin  and fluconazole  was employed.Three hours after the surgery, the patient became feverish (body temperature up to 39.5 degrees centigrade). Emergent blood routine test was normal at that time, with blood samples simultaneously collected for hemoculture. The patient’s conditions deteriorated rapidly. Six hours after PNL, her blood pressure decreased to 80/60 mmHg, indicative of septic shock. The body temperature was as high as 41.0 degrees centigrade, and the heart rate was 110 beats per minute. An abnormal blood cytology was noted  and protein level (0.2 g/L). Since the patient was awake and cooperative, electrophysiological studies were performed. Decreased motor nerve action potential, reduced amplitude of the nerve conduction potentials ) and absent sensory nerve action potential (SNAP) with preserved conduction velocity were found, which pointed to a diagnosis of axonal motor polyneuropathy and myopathy. Combined with the elevated level of CK-MM in the serum, a diagnosis of CIP/CIM was thus established.Two weeks after PNL, the patient turned conscious and partially cooperative to neurological examinations. The proximal motor power of her upper extremities was 1/5 and distal 2/5; the proximal motor power of her lower extremities was 1/5 and distal 2/5. Her muscle tone was reduced symmetrically in the limbs. Sensory tests including light touch, pain and vibration were not affirmatory. Lumbar puncture was performed, showing negative findings in the cerebrospinal fluid (CSF) with regard to cell count  occurring in previously healthy persons. PatientSupportive measures including nutritional interventions, supplement and anti-oxidant therapy, and the application of testosterone derivates, growth hormones and immunoglobulins were proposed to manage the muscle weakness in critically ill patients Figure 30]. Ne. Ne30]. In summary, we reported a case of CIP and CIM as a severe complication of PNL. This case expands our understanding of the complications of PNL and underscores the importance of differentiating CIP/CIM from GBS in case of such patients developing weakness rapidly after the procedure. Diagnosis should be achieved at an early stage and treatment be initiated immediately. Both anti-septic prophylaxis and control of hyperglycemia might be effective for the prevention of CIP or CIM. Avoiding or limiting the use of corticosteroids and NMBAs, as well as EMS and early rehabilitation is also suggested.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Series Editor of this journal.ARDS: Acute respiratory distress syndrome;AST: Aspartate transaminase;IP: Critical illness polyneuropathy;CIM: Critical illness myopathy;CK: Creatine kinase;CMAP: Compound muscle action potential;CSF: Cerebrospinal fluid;DMS: Direct muscle stimulation;ECG: Electrocardiography;EMG: Electromyography;GBS: Guillain-Barré syndrome;ICU: Intensive care unit;IVU: Intravenous urography;PNL: Percutaneous nephrostolithotomy;MOF: Multiple organ failure;SNAP: Sensory nerve action potential;UPJ: Ureteropelvic junction;WBC: White blood cellThe authors declare that they have no competing interests.HL and LMW collected the history of the case and together with HY, RZ and HYL, drafted the manuscript. XBK critically reviewed the manuscript. HLZ finalized the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/36/prepub


Adenosine concentration dependently (2.3–457 μM) platelet aggregation inhibited induced by ADP. Also, adenosine present inhibition of platelet secretion and thrombus formation under flow conditions. The quantitative HPLC analysis revealed significant amounts of adenosine in ripe tomato fruits and its processing by-products. From these results, extracts/fractions of ripe tomato fruits and their processing by-products may be referred to as functional food and functional ingredients containing a compound that inhibits platelet function with a potent preventive effect on thrombus formation, as those that occur in stroke.In seeking the functionality of foodstuff applicable to medicine, ripe tomato fruits were found to show an antiplatelet activity. Therefore, the bioactive compound was isolated, structurally identified, and studied for an inhibitory effects on platelet adhesion, secretion, and aggregation. The concentration of adenosine in ripe tomato fruits (pulp and skin extracts) and its processing by-products (paste and pomace) was determined by reversed-phase high-performance liquid chromatography (HPLC). According to platelet aggregation inhibition induced by ADP, the total extract residual was fractionated by liquid-liquid separation, obtaining aqueous, ethyl acetate and petroleum ether extracts. The aqueous extract was subjected to repeated permeation over sephadex LH-20 and semipreparative TLC. The isolate finally obtained was identified as adenosine on the basis of ESI-MS,  According to the World Health Organization, cardiovascular disease (CVD)  represents about 30% of deaths worldwide , with a The current lifestyle of the population contributes to the development of risk factors for CVD, such as hypertension, diabetes, smoking, and hypercholesterolemia , 4. The In the context of atherosclerosis, platelets can adhere to endothelial cells and contribute to the recruitment of leukocytes involved in the local vascular inflammation , 7. PlatSolanum lycopersicum), fresh or processed , apart from their nutritional value, have been found to provide a cardioprotective effect, both at the endothelial and platelet levels − ion.ESI-MS/MS data was collected using a high resolution hybrid quadrupole (Q) and orthogonal time-of-flight (TOF) mass spectrometer  with constant nebulizer temperature of 80°C. The ESI source and the mass spectrometer were operated in a negative ion mode, and the cone and extractor potentials ware of 10 eV, with a scan range of μL of PBS. The content of adenosine was expressed in mg adenosine/mg dried extract. All samples were filtered through a Millex-LS PTFE filter with 5 μm pore size ; before HPLC analysis, the injection was made in duplicate for 100 μL.Standard solutions of adenosine 3.75 mg/mL for quantification were prepared dissolving 187.5 mg in a volumetric flask of 50 mL . The points of the curve were realized by the dilution of the stock solution between 3.75 and 0.19 mg/mL to produce a sequence of 3.75, 1.88, 0.75 and 0.19 mg/mL in buffer PBS. Extracts of ripe tomato fruits (pulp and skins extracts) and their by-products of processing (paste and pomace extracts) were lyophilized − at m/z 266.7856 [Rt = 7.8 min by HPLC) (1H NMR spectrum of BC was consistent with the structure of adenosine δ (ppm): 8.347 ; 8.133 ; 7.353 ; 5.870 ; 5.44 ; 5.15 ; 4.610 ; 4.136 ; 3.960 ; 3.365 ; 3.549 ; for C10H13N5O4 found following is obtained: C: 44.94 H: 4.90 N: 26.21 and O: 23.95. The data obtained was consistent with previous reports [The BC band was identified as adenosine according to the UV spectrum (by HPLC) . The str reports .P < 0.05). As well as inhibiting the platelet aggregation, the aqueous extract inhibited the platelet secretion in 50 ± 5%.The results of platelet aggregation induced by the agonist ADP with extract, fraction, and adenosine (band C) are presented in μM), in which a concentration of 4.6 μM inhibited 50 ± 12% platelet aggregation (P < 0.05). At the same concentration, it completely inhibited platelet secretion (μM displayed a net lag time of 53 ± 2 s (P < 0.05).The inhibited ADP-induced platelet aggregation of adenosine was concentration dependent   the platelet coverage . Finally, the aqueous extracts (1 mg/mL) of tomato paste and pomace inhibited platelet aggregation ADP induced in 40 ± 3% and 35 ± 1%, respectively, as compared to negative control (P < 0.05) (The inhibition of platelet aggregation ADP-induced by the methanol extract of tomato skins (1 mg/mL) was 40 ± 1% as compared to control ( < 0.05) . r = 0.994. The pulp extracts  showed the highest content of adenosine, while tomato pomace showed the lowest amount. Finally, adenosine content in each extract was positively correlated with the inhibition of platelet function measured by platelet antiaggregant activity  (The adenosine content in ripe tomato fruits and its processing by-products are presented in  < 0.05) .Epidemiological studies have provided evidence of a protective role of healthy diets in the prevention of cardiovascular disease and cancer . Among tin vitro [in vivo [It was observed that tomatoes exert in vitro  and in v[in vivo  antiplat[in vivo . Recentl[in vivo .For extraction and fractioning stages, were fully ripe tomato fruits used showing an optimal industrial quality based on the firmness and soluble solid content . The liqThus, in this study, liquid-liquid separation allowed concentrating antiplatelet activity in the aqueous extract, discarding the presence of compound in ethyl acetate  and petroleum ether extracts  . Such abIn the present study, besides the known platelet antiaggregant activity, it was demonstrated that aqueous extract inhibits platelet function completely: platelet adhesion, secretion, and aggregation.Adenosine at a low concentration showed a potent antiplatelet activity through the inhibition of platelet aggregation and secretion; it also displayed a net lag time in the platelet aggregation assay induced by arachidonic acid, reaching the maximum aggregation rate of >80% at 360 s (data not shown). Moreover it presented inhibition of platelet thrombus formation under flow conditions. Adenosine, which can reach circulating blood from the heart, endothelium, and other tissues, is an endogenous antiaggregating substance that influences the function of circulating platelets by increasing cAMP and cGMP levels .The quantitative HPLC analysis revealed that significant amounts of adenosine were contained in ripe tomato fruits and its processing by-products. Then liquid-liquid separation, the major content of adenosine in aqueous extract, is due to the affinity characteristics of this compound by polar extracts. On the other hand, the lowest adenosine content in tomato paste (rich in tomato pulp) may be due to the heat treatment, and to a lesser extent, because the sterilization stage during the paste-making process affects this compound negatively . Tomato The present study confirms the antiplatelet activity of tomatoes; thus, based on the moisture rate and quantitative analysis of HPLC, 1 kg of fresh tomato contains about 30-times more adenosine than lycopene. So, tomato fruits and their by-products of processing have other bioactive compound, biologically complementing the activities of lycopene . Finally, according to a correlation study, knowing the adenosine content in different extracts is possible to establish the degree of platelet aggregation inhibition. From these results, extracts/fractions of ripe tomato fruits and their by-products of processing may be referred to as functional food and functional ingredients containing a compound that inhibits platelet function with a potent preventive effect on thrombus formation, as those that occur in stroke.Through bioassay guided on the basis of antiplatelet activity, was possible to isolate, identify, and determine a bioactive compound with a complementary biological activity to that of the lycopene. The antiplatelet activity is specific for adenosine, so that, according to its content, it is possible to establish the degree of such activity in the different types of extracts. Based on the present results, extracts of ripe tomato fruits and their processing by-products due to their adenosine content may be used as functional ingredients adding antiplatelet activities to processed foods which may be supportive in the primary prevention of CVD.Supplementary video 1: Adenosine Inhibit Collagen-Induced Platelet Thrombus Formation under Flow Conditions.Supplementary video 2: Collagen-induced platelet thrombus formation under flow conditions.Supplementary video 3: Aqueous Extract Inhibit Collagen-Induced Platelet Thrombus Formation under Flow Conditions.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Multiple myeloma (MM) is a low proliferative tumor of postgerminal center plasma cell (PC). Centrosome amplification (CA) is supposed to be one of the mechanisms leading to chromosomal instability. Also, CA is associated with deregulation of cell cycle, mitosis, DNA repair and proliferation. The aim of our study was to evaluate the prognostic significance and possible role of CA in pathogenesis and analysis of mitotic genes as mitotic disruption markers.A total of 173 patients were evaluated for this study. CD138+ cells were separated by MACS. Immunofluorescent labeling of centrin was used for evaluation of centrosome amplification in PCs. Interphase FISH with cytoplasmic immunoglobulin light chain staining (cIg FISH) and qRT-PCR were performed on PCs.P = 0.019).Based on the immunofluorescent staining results, all patients were divided into two groups: CA positive (38.2%) and CA negative (61.8%). Among the newly diagnosed patients, worse overall survival was indicated in the CA negative group (44/74) in comparison to the CA positive group (30/74) . Gene expression was significantly down-regulated in newly diagnosed in comparison to relapsed patients in the following genes: AURKA, AURKB, CCNB1, CCNB2, CETN2, HMMR, PLK4, PCNT, and TACC3 (P < 0.05).Gene expression was significantly down-regulated in the CA positive group in comparison to CA negative in the following genes: Our findings indicate better prognosis for CA positive newly diagnosed patients. Considering revealed clinical and gene expression heterogeneity between CA negative and CA positive patients, there is a possibility to characterize centrosome amplification as a notable event in multiple myeloma pathogenesis. Centrosomes are small organelles composed of two cylindrically shaped centrioles surrounded by pericentriolar material in a normal mitotic cell. The centrosome function is to direct mitotic bipolar spindles in a process that is essential for accurate chromosome segregation during mitosis ,2. AfterA vast number of solid and hematologic cancers harbor centrosome amplification ; most ofConversely, there is evidence that the centrosome contributes to cell-cycle regulation and checkpoints ,16-20. BMM is a low proliferative tumor of postgerminal center PC. Normal PCs are arrested in the G1 phase of the cell cycle. Malignant PCs have a proliferation rate that increases from the early to the advanced stage of plasma cell dyscrasia -25 and tPreviously published data about clinical implication of CA was reported based on the so-called centosome index (CI) . In factAlthough clinical implication of centrosome amplification in MM is still unclear, it is obvious that the role of CA as well as CA formation in MM differ from other malignancies. We anticipate that such differences are based on specificity of malignant transformation in MM genesis. The aim of our study was to evaluate prognostic significance and possible role of centrosome amplification in pathogenesis and analysis of mitotic genes as mitotic disruption markers.A total of 173 MM patients enrolled in University Hospital Brno, Czech Republic, University Hospital Olomouc, Czech Republic and University Hospital Bratislava, Slovakia, were included in this study. The patients’ baseline characteristics are summarized in Table PCs visualization with immunoglobulin light chain staining (cIg) and immunofluorescent labeling of centrin as an integral centrosome protein for centrosome copy number determination, was performed as described previously ,34. BrieCytospin slides for immunolabeling detection of CA in PCs are prepared as follows: approximately 100,000 BMMC are placed on a slide and air dried for 24 hrs at room temperature. Then, PCs are fixed in ice-cold methanol for 5 min at room temperature. Cytoplasmic membrane permeabilization is done by Triton X-100 0.2%  for 1 minute at 37°C. After that, slides are placed in PBS for 10 min using gentle agitation. To prevent non-specific binding, blocking buffer  is added to each slide and incubated for 20 min in the wet chamber at 37°C. After incubation, the blocking buffer is poured off by gentle agitation during 10 min. After that, 20 μl of diluted (3 : 1000) primary antibody  is applied to each slide. The slides are incubated for 1 hr in the wet chamber at 37°C. To wash out unspecific antibody binding, slides are washed 3 times with phosphate buffered saline (PBS) for 5 min each using gentle agitation. The secondary goat anti-rabbit IgG antibody (1.5 : 1000) is applied  and incubated under the same conditions for 45 min. Another step of washing is done again 3 times . Further immunolabeling on cytospin slides is done, using immunoglobulin light chain staining, according to the procedure described previously ,32,34.FISH was performed as a part of routine diagnostic procedure according to the previously described protocol . The folTotal RNA was isolated using QIAGEN RNeasy Mini Kit. RNA isolation and purification were described previously . RNA wasTotal RNA with purity ratio 260/280 >1.7 and integrity (RIN) >7.5  was used for further analyses.Quantitative RT-PCR was focused on a chosen list of genes, according to their role in normal centrosome duplication process. qRT-PCR was performed using the Applied Biosystems platform. Input of 100 ng of high-quality total RNA was reverse transcribed using High Capacity cDNA Reverse Transription Kit and preamplified with TaqMan PreAmp MasterMix Kit . Expression of each gene was evaluated in a duplex reaction by TaqMan Gene Expression Assays and GAPDH as an internal control on 7500 Real Time PCR System. Relative fold change of expression for each gene was calculated using the ΔΔCt method.P-values below 0.05 were considered statistically significant.For continuous variables, nonparametric Kruskal-Wallis or Mann–Whitney tests were used. For discrete variables, chi-squared test for independence of Fisher’s exact test was used. The overall survival (OS) calculated from the date of diagnosis and survival rates were estimated using the Kaplan-Meier method. Differences of survival among subgroups of patients were compared using the log-rank test. Cox proportional hazards models were used to assess the association of several prognostic factors with OS. All patients involved in this study were stratified into two subgroups (CA negative and CA positive) based on centrin immunofluorescent staining results. Frequencies of patients among subgroups were as follows: 38.2% (66/173) in CA positive, 61.8% (107/173) in CA negative, respectively.P = 0.29) as well as ISS staging (P = 0.46). Comparison of biochemical parameters showed significant differences in hemoglobin and creatinine level between CA positive and CA negative groups , translocation t, 1q21 gain and hyperdiploidy detected by FISH in these two subgroups  . Univariate Cox proportional hazards survival model with one explanatory variable showed worse prognosis for CA negative group . All disease-related deaths occurred in two years after the diagnosis and were significantly higher in the CA negative group (7/44) than in the CA positive (0/30) group of patients (P = 0.037).To determine the prognostic impact of patients in defined subgroups, we compared their overall survival. Although statistical significance was not reached for the whole studied cohort (data not shown), significant differences were observed in newly diagnosed patients (74/139). Worse OS was indicated in CA negative patients (44/74) in comparison with CA positive patients (30/74)  Figure . None ofTo further characterize the prognostic significance of CA multivariate Cox proportional hazards survival model was used Table . The varAURKA, AURKB, CCNB1, CCNB2, HMMR, PLK4, PCNT, TUBG1, TACC3, and CDK2) associated with centrosome structure/function were selected for qRT-PCR analyses. We found significant differences in relative quantification coefficient R of AURKB, PLK4 and TUBG1 genes that were down-regulated in the CA positive group in comparison with CA negative patients (p < 0.05)   was significantly different between hyperdiploid and non-hyperdiploid myeloma patients  is associated with poor prognostic genetic subtypes and portends short survival ,33. On tWe suggest that CA presence can be explained as heritage from myeloma progenitors and their mitotic checkpoint alteration, whereas gene expression changes can occur in numerous physiological and/or pathological processes. This statement is in concordance with other findings in expression changes of genes associated with centrosome structure/function. Expression of the above mentioned genes was different not only in CA subgroups or related to ploid category but it was also different in newly diagnosed and relapsed patients.A cancer cell may be derived from a mature progenitor cell that has acquired stem cell properties, i.e. self-renewal and immortality. In this case, the oncogenic event(s) enable(s) the cell(s) to self-renew. It is possible that some events, such as oncogenic targeting of the centrosome, trigger proliferation, survival and self-renewal at the same time and can target indifferently committed progenitors .Although most of the multipolar divisions that occur in tumors probably reflect non-productive events, an occasional division might give rise to progeny with a genetic constitution that favors survival in a changing physiological environment. Selective pressure might arise, for instance, through increasing hypoxia or nutritional deprivation in a growing tumor mass, or through the presence of a chemotherapeutic drug . Damage Obviously, our findings need to be confirmed and validated on a larger external cohort of patients. We believe that future investigation of centrosome amplification will help to refine the broad prognoses offered by current established systems and even sub-stratify them.The authors reported no potential competing interest.ED and FK were the principal investigators and take primary responsibility for the paper. RH, JM and ZS recruited the patients. ED, FK, LK, PN, and SS performed the laboratory work for this study. II and JJ participated in the statistical analysis, RH and PK coordinated the research. ED and FK wrote the paper. All authors read and approved the final manuscript.Comparison of incidence of chromosomal abnormality in CA groups of patients.Click here for fileCox proportional hazards survival model with combination of centrosome amplification and one another explanatory variable.Click here for file


Salmonella enterica serotype Typhi strains with a nonclassical quinolone resistance phenotype  associated with a nonsynonymous mutation at codon 464 of the gyrB gene. These strains, not detected by the nalidixic acid disk screening test, can result in fluoroquinolone treatment failure.We report  Salmonella enterica serotype Typhi  remains a major health problem in the developing world  (MIC >256 μg/mL) and show decreased susceptibility to Cip (CipDS)  have emerged and become endemic on the Indian subcontinent and in Southeast Asia   and those from the antibiogram committee of the French Society for Microbiology   have not been reevaluated  mutations in codons 83 or 87 of gyrA are uniformly NalR. However, recent reports have indicated that this approach cannot identify the newly described Salmonella Typhi isolates that are Nal susceptible –CipDS for which mechanisms of resistance are not linked to mutations in gyrA .Typhoid fever caused by Salmonella Typhi isolates in France are referred to the FNRC-Salm, and almost all are acquired abroad, mainly in Africa and Asia. Until 2009, CipDSSalmonella Typhi was monitored with the 30-μg Nal screening test. A total of 685 Salmonella Typhi isolates collected during 1997–2009 were reanalyzed to identify NalS–CipDSSalmonella Typhi isolates. The scattergram correlating the zone diameters around the 5-μg ciprofloxacin disk with those of the 30-μg Nal disk showed 4 subpopulations, which were labeled A (554 isolates), B (11 isolates), C (119 isolates), and D (1 isolate) . In subpopulation A, 75 isolates had wild-type QRDR sequences, whereas 2 isolates had a gyrB mutation at codon 465 leading to amino acid substitution Gln to Leu. Their Nal MICs were 2 and 4 μg/mL, respectively, and those of Cip were 0.04 μg/mL and 0.08 μg/mL, respectively. Notably, both isolates were acquired in Mexico during 1998 and 2009, respectively. In subpopulation C, the lowest MIC values for Cip (0.06 μg/mL) were associated with a mutation at codon 87 of the gyrA gene, whereas MICs did not increase with the additional mutation in the parE gene. Subpopulation D consisted of 1 isolate, highly resistant to ciprofloxacin, which was acquired by a traveler in India in 2004. This isolate contained 2 NS mutations in the gyrA gene and 1 in the parC gene.In France, laboratory surveillance of typhoid fever infections is performed by the FNRC-Salm through its network of ≈1,500 hospital and private clinical laboratories. Almost all isolate) . The cha<16 μg/mL; resistance, >32 μg/mL). Of the 11 isolates, 8 (from 7 patients) had a ciprofloxacin MIC >0.125 μg/mL and were thus classified as CipDS isolates. We were able to review the medical records of 2 patients infected with a NalS–CipDS isolate. One patient (isolates 08-7675 and 09-1986) relapsed 15 days after completion of the treatment  , qepA, and aac(6′)-Ib-cr were not detected by PCR  subtyping . Although Cooke et al. (S–CipDS represented 11.6% (49/421) of Salmonella Typhi isolated in England, Scotland, and Wales during 1999–2003, while Lynch et al. (Salmonella Typhi isolates identified in the United States during 1999–2006. Epidemiologic data were available for 39 isolates in the British study, 18 of which were acquired in India, 8 in Pakistan, and 4 in Bangladesh (Eleven isolates of subpopulation B were categorized as susceptible to Nal by determining MICs and by using CLSI breakpoints (susceptibility, GE types . AccordiGE types . In our S–CipDSSalmonella Typhi isolates originating from Asia comprise ≈1% of Salmonella Typhi isolates in France but are more prevalent in the United States and the United Kingdom. The NS gyrB mutation at codon 464 was found exclusively in NalS–CipDS isolates; however, the effects of this mutation need to be formally demonstrated by site-directed mutagenesis. Furthermore, the involvement of an efflux system, such as AcrAB-TolC and OqxA, or the qnrC gene, have not been investigated and cannot be excluded.NalR screening test should no longer be recommended and ciprofloxacin drug MICs should be determined for all Salmonella Typhi isolates instead. There is also a clear need to reevaluate the clinical breakpoints for this pathogen.Whatever the molecular mechanism of resistance of such strains, the main concern is detection of such isolates in clinical practice to prevent fluoroquinolone treatment failures. Consequently, the Nal


Entamoeba histolytica is a protozoan parasite that presents a risk to the health of millions of people worldwide. Due to the existence of different clinical forms caused by the parasite and also different virulence levels presented by one strain, one would expect differences in the profile of gene transcripts between virulent and nonvirulent cultures. In this study we used the differential display to select gene segments related to invasiveness of amoeba. One Brazilian strain of E. histolytica in two conditions, able or not to cause lesions in experimental animals, was used. RNA from this strain, was used to study the differential expression of genes. 29 specific gene fragments differentially expressed in the virulent strain were selected. By real-time PCR, six of these genes had confirmed their differential expression in the virulent culture. These genes may have important roles in triggering invasive amoebiasis and may be related to adaptation of trophozoites to difficulties encountered during colonization of the intestinal epithelium and liver tissue. Future studies with these genes may elucidate its actual role in tissue invasion by E. histolytica generating new pathways for diagnosis and treatment of amoebiasis. Entamoeba histolytica, the protozoan responsible for amoebiasis, a disease that affects millions of people worldwide [E. histolytica strain and the host immune response [E. histolytica and in different culture conditions is extremely important for a better understanding of the biology of this parasite since give us data to support the participation of new and already known factors on its virulence. In this context, the purpose of this study was to identify genes differentially expressed in trophozoites of the same strain of E. histolytica under different virulence conditions.orldwide , 2, presresponse . Among fresponse . Recent response –7. Differesponse , like paresponse  and prolresponse , were alE. histolytica [The strain ICB-CSP (CSP), previously identified as tolytica , was cho6) were inoculated in the left lobe of the liver of hamsters (Mesocricetus auratus). After 24 hours, the animals were sacrificed and liver fragments were grown in culture medium for reisolation of trophozoites. This strain was renamed as reisolated CSP (CSP-R).To activate the virulence, the trophozoites (1 × 106 cells of strains CSP in culture and CSP-R), isolated directly from hamsters liver. The RNA was resuspended in 0.01% DEPC water, quantified by spectrophotometry, and stored in a freezer at −80°C until use. Three microliters of RNA treated with deoxyribonuclease I  was used for cDNA synthesis. The reaction contained 25 ng/μL oligo (dT)12–18, 500 μM each dNTP, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, and 200 units of reverse transcriptase  in 50 mM Tris-HCl buffer (pH 8.3) in a final volume of 10 μL. The reaction was carried out at 42°C for 50 minutes, as negative control samples were also prepared without addition of reverse transcriptase. Differences in expression levels between CSP and CSP-R were analyzed using the RNA differential display as previously described [Total RNA was extracted  from trophozoites , in TAE buffer (Tris-acetate-EDTA), under an electric field of 100 volts. The gels were stained with silver nitrate [2, 0.1% Triton X-100. This mixture was heated at 95°C for 20 minutes. The cDNAs extracted from the gel were used in a reamplification PCR using 10 mM Tris-HCl pH 8.4 buffer containing 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200 μM each dNTP, 0.6 μM of one primer, and 1.0 unit of Taq DNA polymerase . The program used was similar to that described above excluding the initial cycle of annealing at 72°C. The products were loaded on a 1% agarose gel stained with ethidium bromide (0.5 μg/mL) and purified from the gel using the GFX kit (Amersham Pharmacia Biotech Inc.) according to manufacturer's instructions. The purified cDNA was cloned into pGEM-T easy vector  and transformed into Escherichia coli, strain DH5α. Positive clones were selected using the system ampicillin/IPTG/X-Gal [PCR was carried out in 10 mM Tris-HCl pH 8.4 buffer containing 50 mM KCl, 1.5 mM MgCl primers , and 0.1http://www.ncbi.nlm.nih.gov/BLAST) and Sanger Institute (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/e_histolytica) using the Basic Local Alignment Search Tool program [Recombinant plasmids were isolated using Wizard Plus SV kit minipreps  and submitted to sequencing reaction. The sequencing reaction was performed according to the method previously described . Reactio program .μL containing Power SYBR Green PCR Master Mix , 0.45 μM each primer, and 1 μL of cDNA. Amplification conditions consisted of 40 cycles of 95°C/15 s, 60°C/45 s, and 72°C/30 s. Reactions were carried out in duplicate. The dissociation stage was added to the program for which the analysis of specific amplification could be made after completion of reactions. For relative quantification of amplification products the method 2−ΔΔCt was used [The Kit SYBR Green PCR Master Mix  was used. The primers for all sequences were designed using Primer Express V2.0 program  to generate fragments between 70 and 190 bp. All primers were previously tested in conventional PCR using the plasmids containing the cloned fragment as a positive control. As internal control, the expression levels of the actin gene was determined . The reawas used .http://www.cbs.dtu.dk/services/. Only the sequence BH10.9 showed signal peptide prediction. The genes showed differential expression in the range of 12.1- to 339-fold more expressed in the virulent culture (CSP-R) compared to the nonvirulent culture (CSP) (The CSP strain had its virulence activated after the second passage through hamster liver. The lesions produced belong to the class IV as previously defined . Only twre (CSP) .E. histolytica is a pathogenic organism in which its virulence varies according to environmental conditions [It is well established that nditions . Therefonditions –23.E. histolytica presenting different virulence conditions [E. histolytica with high aggressiveness to experimental animals. This strain had its virulence attenuated by prolonged cultivation becoming unable to injury tissues. Its virulence was activated by inoculation into hamster liver. As the attenuated and activated isolates were taken from axenic culture, the differences found in the strain invading tissues can reveal new molecules involved in amoebic pathogenicity, in addition to confirmation of virulence genes found by other authors. For gene expression evaluation, the RNA differential display technique was used to be simple and allow the analyses of a large number of different transcripts from a small amount of RNA [E. histolytica (CSP-R) was selected. From 13 sequences that were successfully reamplified, 12 had their level of expression confirmed by semiquantitative RT-PCR. However, only 6 had their expression level successfully analyzed by RT-qPCR.Alterations in the expression pattern of molecules related to virulence may help to define invasiveness markers. Previous research has demonstrated differences in gene expression in trophozoites of nditions , 24–26. t of RNA . 29 segmSYBR green, a nonspecific fluorescence marker that binds to double-stranded DNA, was used in RT-qPCR. The SYBR contains in its structure a quaternary nitrogen positively charged, responsible for binding to DNA. We speculate that either the sequence composition or the amount and chemical characteristics of ions present in the samples could have interfered with the SYBR binding to the DNA. As a result, the fluorescence intensity would decrease, making detection of low expressed genes impractical. This may have occurred with the nonamplified fragments in RT-qPCR in this study.Leishmania mexicana and Trypanosoma, being an essential enzyme for recycling membrane and endocytosis [E. histolytica. Another gene more expressed in CSP-R is the Cdc48-like protein (fragment BH18.36). This gene is also more expressed in E. histolytica stimulated by type I collagen and calcium ATPase activity presenting and participating in the processes of endo- and exocytosis [E. histolytica is known for its high content of vesicles containing aggressive molecules such as lysozyme, pore-forming peptides, and proteinases.One of the more expressed sequences of virulent culture is that of coding for acid phosphatase gene (fragment BH4). The acid phosphatase has been described in parasites such as ocytosis , and mayocytosis . E. histTaking into account that it was evaluate transcripts of the same strain, capable (CSP-R) and unable (CSP) to cause lesions, the increased expression levels of genes encoding proteins such as acid phosphatase and EhCdc48-like, which are important elements in the regulatory mechanism of endocytosis, fusion of lysosomes, and recycling of membrane, suggest that these proteins may have important role in tissue invasion by the amoebae.Four distinct sequences encoding hypothetical proteins  had also confirmed the differential expression, all of them more expressed in the virulent culture. One of these proteins had signal peptide, should be secreted, and may have a more direct action in the process of tissue damage in the host.Differences in gene expression presented here as well as those described by others may be related to adaptation of trophozoites due to difficulties during the colonization of the intestinal epithelium and liver tissue. Future studies of these genes may elucidate its actual role in amoebic tissue invasion, generating new pathways for diagnosis and treatment of amoebiasis.


Purpose. Contrast-induced nephropathy (CIN) can contribute to acute kidney injury (AKI) in patients undergoing endovascular aortic aneurysm surgery. We evaluated the incidence of AKI together with the evolution of early biomarkers of renal injury in patients receiving bicarbonates or NaCl 0.9%. Methods. This study involved endovascular aortic aneurysm surgery patients. Group A (n = 17) received bicarbonates 3 mL/kg/h for 1 h before the procedure and then 1 mL/kg/h until 6 h after surgery, whereas group B (n = 17) received NaCl 0.9% using the same protocol. Biomarkers of renal injury from urine (interleukin-18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL), N-acetyl-β-D-glucosaminidase (NAG), and kidney injury molecule 1 (KIM-1)) and blood  were measured at baseline and 3, 24, and 48 h postoperatively. Results. AKI occurred in 1 patient (2.9%), in the bicarbonates group. IL-18, NAG, NGAL, and KIM-1 significantly rose in both groups after the surgery. There was a greater rise in NGAL and IL-18 after 3 h in the bicarbonates versus NaCl 0.9% group: 1115% versus 240% increase (P = 0.03) and 338% increase versus 1.4% decrease (P = 0.01). Conclusions. Despite significant elevation in biomarkers of renal injury, we demonstrated a low rate of AKI following endovascular aortic surgery. The endovascular approach for the repair of aortic aneurysm is an interesting alternative to the open repair approach because of its associated reduced 30-day morbidity and mortality. This approach, however, carries its own risks such as renal complications –5.Renal complications occur with variable incidence, ranging from 5.5 to 20% –6. They Many strategies have been developed in an attempt to prevent CIN. Among these, an intravenous volume expansion that covers the pre-, per- and postcontrast media exposure is the only established way –13. N-acWhat type of infusion to use to achieve intravenous volume expansion is not known –29. By iβ-D-glucosaminidase (NAG), kidney injury molecule 1 (KIM-1) blood NGAL, and cystatin C, have been studied, and their evolution has been linked to the subsequent development of AKI [Furthermore, serum creatinine is typically used for the diagnosis of AKI, but it is an insensitive and unreliable biomarker during acute changes in kidney function . More set of AKI –39. The incidence of renal complications during endovascular aortic aneurysm repair varies in a fourfold proportion and furthermore may be modified by the hydration protocol used; therefore, their real incidence is not known. The primary goal of this pilot study was to evaluate the true incidence of AKI in this population of patients and to compare the evolution of numerous early biomarkers of renal injury before and after contrast media exposure in patients receiving either sodium bicarbonate or sodium chloride for intravascular volume expansion during endovascular aortic aneurysm repair. ISRCTN39325111) that took place between March 2009 and March 2011. The incidence of postoperative AKI, defined by an elevation in serum creatinine beyond 50% of baseline within 48 h (acute kidney injury network [β-D-glucosaminidase (NAG), interleukin-18 (IL-18), cystatin C (cysC), and kidney injury molecule-1 (KIM-1). The need for dialysis and mortality within 30 days of the surgery was also recorded.This is a prospective randomized controlled pilot study , was evThe study protocol was approved by our local ethic committees . Over a two-year period, patients ranging from 18 to 85 years of age, and presenting for elective endovascular aortic repair, were approached by a member of the research team at Montréal Hôtel-Dieu (CHUM) and Maisonneuve-Rosemont hospitals . Exclusion criteria included patient refusal, exposure to contrast media within 14 days prior to surgery, acute renal failure on the day of surgery, defined as serum creatinine 50% above normal patient values, patients who were renal transplant recipients, and patients on dialysis for chronic renal failure. All patients signed a written informed consent.Patients were randomly assigned based on a computer-generated list. Patients in group A were assigned to receive an infusion of sodium bicarbonate, whereas patients in group B received a sodium chloride (NaCl 0.9%) infusion. All patients received NAC. The randomization list was generated by the pharmacy which then supplied both solutions in an unidentifiable infusion bag to a blinded member of the research team.Immediately after arriving in the operating room, study participants received their first dose of NAC, which was administered as an intravenous (i.v.) bolus over 15 minutes. This method is slightly different from the most common practice, however, can be used, and its efficacy has been demonstrated, particularly when patients cannot be prepared because of urgent procedure or lack of time . The infAt the induction of general or spinal anesthesia, approximately 1 h before the contrast media was introduced, the study infusion were administered according to a protocol that is, used in most studies on bicarbonates for CIN prevention , 23, 26:The attending anesthesiologist, who was blinded to patient group, was instructed to give crystalloids (Lactated Ringer), colloids (6% hydroxyethyl starch (HES) 130/0.4) or blood products according to clinical judgement to cover for insensible and blood losses. The contrast media administered to the patients were iodixanol , a nonionic iso-osmolar opacifying agent. Mannitol and furosemide use was not permitted. Diuretics were routinely withheld on the day of surgery. Nonsteroidal anti-inflammatory agents use was prohibited during the entire perioperative period.Information on demographics and comorbid conditions was obtained from the patients and their medical records. CIN risk score was then calculated for each patient. This risk score, as developed by Mehran et al.  for patiDetailed medication history was recorded. Recorded procedural data included the occurrence of intraoperative hypotension, which was defined as systolic blood pressure below 100 mm Hg for at least 30 minutes or necessitating the use of vasopressors to be maintained above 100 mm Hg, blood losses, total volume of crystalloids, colloids, blood products, and total volume of contrast media administered.Samples were collected to evaluate the evolution of the following biomarkers: urinary NGAL, IL-18, NAG, KIM-1 serum NGAL, and cysC. Urinary and blood specimens were collected after the induction of anesthesia and 3 h, 24 h, and 48 h after exposure to contrast media. Serum creatinine was collected at the same time points. Samples were kept frozen at −80°C up to the time of the analysis. NGAL in serum and urine was assayed using Rapid ELISA kit from Antibody Shop A/S . IL-18 was measured in urine by using quantitative ELISA assay from Medical & Biological Laboratories Co. Ltd. . KIM-1 and cystatin C were measured using Quantikine Human TIM-1/KIM-1/HAVCR and Quantikine Human cystatin C ELISA kit, respectively, from R&D Systems . Urinary NAG activity was assayed manually using NAG assay from BioQuant . Creatinine was measured on Architect C16000, Abbott Diagnostics .t- and chi-squared tests, respectively. Kruskal-Wallis and Mann-Whitney U tests were used to analyze ordinal and nonnormally distributed continuous data.Data were collected using Microsoft Excel and were analyzed using IBM SPSS Statistics 19. All continuous variables were first analyzed with a one sample Kolmogorov Smirnov test to determine normality. Demographic and perioperative continuous and categorical data were analyzed using Student's t-test. Comparison between study groups for the percent change in each biomarker at each time point was analyzed with a Student's t-test. A P value < 0.05 was considered statistically significant.According to each study group, the incidence of postoperative AKI was analyzed as a categorical data with a Fisher's exact test. Serum and urinary levels for each of the biomarkers were compared to their baseline levels by calculating the percentage of change that was analyzed with a Student's From March 2009 to March 2011, 57 eligible patients were asked to participate in the research project. Of these, six were not randomized because the planned surgery also included vascular procedures on vessels other than the aorta  and iliac stenting (3)). Seven patients refused to participate, and ten patients were excluded: age >85 years old (2), chronic kidney failure on dialysis (2), patient preference for an open repair (3), and exposure to contrast media in the last 14 days (3). A total of 34 patients were included, 17 randomized to group sodium bicarbonate and 17 to group sodium chloride . All patP = 0.31). Baseline levels for creatinine, cysC, serum and urinary NGAL, IL-18, NAG, and KIM-1 were comparable between groups  of the patients. AKI occurred in 1 patient in the sodium bicarbonate group (5.9%), compared to no patient in the sodium chloride group (0%): difference 5.9%, 95% CI of −5.3% to 17.1%, P = 0.03).Overall, most biomarkers of renal injury and cysC increased from baseline after exposure to contrast media, with this change being statistically significant only for urinary IL-18 at 24 and 48 h, NAG, and KIM-1 . ConversP = 0.03) and 338% increase versus 1.4% decrease (P = 0.01), respectively. This difference was not found at other time points.There was a significantly greater rise in urinary NGAL and IL-18 after 3 h in the bicarbonate versus sodium chloride group: 1115% versus 240% increase .Our results also show an increase in all biomarkers of renal injury following exposure to contrast media. This increase was significant for NAG and KIM-1 as early as 3 h after exposure, whereas it took 24 h for IL-18 and urinary NGAL to increase significantly. This can be attributed to their different kinetics , 39. These biomarkers are produced by renal tubular cells and are highly specific for renal tubular injury –39. ThisThe low rate of AKI observed in this study could also be due to the fact that we stopped assessing creatinine 48 h postoperatively. Although creatinine typically starts to rise within the first 24 h in most cases of CIN , it is pFurthermore, some of these biomarkers might be too sensitive, without being clinically relevant. Exposure to contrast media has caused an increase in urinary NAG excretion, without leading to AKI –39. The rise in these biomarkers was higher in the bicarbonates group for urinary IL-18 and NGAL 3 h after exposure to contrast media. A greater rise in these specific biomarkers has shown to be an accurate predictor of CIN –34, and This study could not confirm the role of the early biomarkers of renal injury on the prediction of postoperative AKI after endovascular aortic surgery. However, it would be interesting to measure their role in a larger, definitive trial evaluating the impact bicarbonates have on the incidence of postoperative AKI following endovascular aortic aneurysm repair. Given the very low incidence of postoperative AKI demonstrated in this trial, such a study would require 2850 patients per group to have the power of detecting a 30% difference between groups.This pilot study was limited by the small number of patients recruited and by the fact that we stopped assessing for serum creatinine after 48 h. The results of this pilot study indicate a low rate of postoperative AKI following endovascular aortic surgery in a low risk population, despite significant elevation in early markers of renal injury. Their role in the prediction of postoperative AKI has not been substantiated in this study and should be evaluated in a much larger, definitive study determining the impact of bicarbonates on postoperative AKI after endovascular aortic surgery.


Approximately 90 haemoglobinopathies have been identified that result in abnormally high oxygen affinity. One of these is haemoglobinopathy York (HbY), first described in 1976. HbY causes an extreme leftward shift of the oxygen dissociation curve with the P50 value changing to 12.5 - 15.5 mmHg , indicating that approximately half of the haemoglobin is not available as oxygen carrier. Patients with haemoglobinopathies with increased oxygen affinity could suffer from the risk developing ischaemic complications due to a lack of functional oxygen carriers. This is, to best of our knowledge, the first case report on a patient with HbY published in connection with anesthesia.A 42-year-old female with a severe headache and Glasgow coma scale (GCS) of 15 was admitted to the neurosurgical intensive care unit with a ruptured, right sided ICA aneurysm with consecutive subarachnoid haemorrhage . The medical history of the patient included an erythrocytosis (Hb 17.5 g/dl) on the base of a high-oxygen-affinity haemoglobinopathy, called Hb York (HbY). With no time available to take special preoperative precautions, rapid blood loss occurred during the first attempt to clip the aneurysm. General transfusion procedures, according to the guidelines based on haemoglobin and haematocrit values, could not be applied due to the uncertainty in the oxygen carrier reduction. To maintain tissue oxygen supply, clinical indicators of ischaemia were instead utilized to gauge the appropriate required blood products, crystalloids and colloids replacements. Despite this, the patient survived the neurosurgical intervention without any neurological deficit.Family members of patients with HbY (and other haemoglobinopathies with increased oxygen affinity) should undergo clinical assessment, particularly if they are polycythaemic. If the diagnosis of HbY is confirmed, they should carry an "emergency anaesthesiology card" in order to avert perioperative risks arising from their "hidden" anemia. Haemoglobinopathy is defined as a genetic defect that results in an abnormal structure of one of the globin chains of the haemoglobin molecule. It is the most frequently occurring monogenic disorder . To dateApproximately 90 haemoglobinopathies have been identified that result in abnormally high oxygen affinities. The first was reported in 1966 on an 81-year-old patient who presented with polycythemia . Decreas6/μl.A 42-year-old female with a severe headache and Glasgow Coma Scale (GCS) of 15 was admitted from a regional hospital close to our neurosurgical intensive care unit. Digital subtraction angiography showed a ruptured, right-sided ICA aneurysm near the bifurcation followed by a subarachnoid haemorrhage . A neurological examination revealed no deficits. The medical history of the patient included obesity (a body mass index of 29), Hashimoto thyroiditis, and an erythrocytosis (Hb 17.5 g/dl), in addition to the HbY. The diagnosis of HbY was initially performed at the University of Ulm (Germany) in 2002 through molecular genetic analysis, subsequent to the exclusion of alternative explanations for erythrocytosis. Preoperative laboratory findings showed a haematocrit of 54%, Hb 17.5 g/dl, reticulocytes 25/1000 and erythrocytes 6.2 x 102) of 0.5 and a minute ventilation (MV) of 110 ml/min/kg. Nimodipine was continually administered at a rate of 1 mg/h. Anaesthesia was maintained with Propofol (8 mg/kg/h) and Remifentanyl (0.25 μg/kg/h). The operation was started and progressed uneventfully until the surgeon tried to dissect the basis of the aneurysm, which led to severe bleeding. Several temporary clips were required to stop the haemorrhage (approx. 1600 ml). Subsequent to volume resuscitation with 1000 ml Ringer's solution and 500 ml of volume expander  130/0.4, the haemoglobin value was 10.8 g/dl. During the bleeding interval, no S-T segment changes were observed. However, typical signs of rapid fluid loss, such as a moderately increased heart rate and a temporary drop in blood pressure, were present. Systolic blood pressure was always kept between 120 mmHg and 140 mmHg using noradrenalin in varying dosages (max. 0.25 μg/kg/min). Meanwhile, the patient received 6 units of packed red blood cells (PRBC) and further fluid substitutions.After induction of anaesthesia with propofol (2 mg/kg), remifentanyl (0.5 μg/kg/min) and rocuronium (70 mg), an arterial and a central-venous catheter were placed in addition to two peripheral intravenous 17 G catheters. The lungs were ventilated with a fraction of inspired oxygen  was identified by DNA sequencing, which indicated an autosomal dominant inherited point mutation of codon 146, where adenosine was exchanged for cytosine (CAC ⇒ CCC). The mutation causes a change from histidine to proline at position 146 in the β-globin chain. The mutant globin has approximately twice the normal affinity for oxygen [2 and thus cannot serve the physiological function of oxygen delivery. As a consequence of decreased tissue oxygen supply, erythropoietin production in the kidneys is upregulated, resulting in erythrocytosis. Preoperative laboratory findings confirmed an usual compensatory mechanism for chronic but moderate tissue hypoxia.The patient's history of haemoglobinopathy begun when a general practitioner suspected polycythaemia vera as an underlying cause for a chronic erythrocytosis. Common causes, including cigarette smoking, psychosocial stress, chronic residence at high altitudes and chronic lung disease, were excluded. Finally, HbY  should undergo clinical assessment, particularly if they are polycythaemic. If the diagnosis of HbY is confirmed, they should carry an "emergency anaesthesiology card" in order to avert perioperative risks arising from their "hidden" anaemia. It should be emphasized that general transfusion guidelines are not applicable to individuals with this rare condition. Therapeutic attempts at decreasing the abnormal oxygen affinity using bezafibrates are still being investigated. Venesection seems to only be sensible in case of repetitive thrombotic events, but the risk of an ischaemia is always present in these patients.Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.EM: Preparation of the manuscript and involvement in the case. TJ: Anaesthesiologist involved in the case. NG: Substantial hematological background and preparation of the manuscript. MW: Preparation of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/12/19/prepub


Bromelain (Br) is a cysteine peptidase (GenBank AEH26024.1) from pineapple, with over 40 years of clinical use. The constituents mediating its anti-inflammatory activity are not thoroughly characterized and no peptide biomarker exists. Our objective is to characterize Br raw material and identify peptides in the plasma of Br treated mice. After SDS-PAGE in-gel digestion, Br  peptides were analyzed via LC/MS/MS using 95% protein probability, 95% peptide probability, and a minimum peptide number = 5. Br spiked mouse plasma (1 ug/ul) and plasma from i.p. treated mice (12 mg/kg) were assessed using SRM. In Br raw material, we identified seven proteins: four proteases, one jacalin-like lectin, and two protease inhibitors. In Br spiked mouse plasma, six proteins  were identified. Using LC/MS/MS, we identified the unique peptide, DYGAVNEVK, derived from FB1035, in the plasma of i.p. Br treated mice. The spectral count of this peptide peaked at 6 hrs and was undetectable by 24 hrs. In this study, a novel Br peptide was identified in the plasma of treated mice for the first time. This Br peptide could serve as a biomarker to standardize the therapeutic dose and maximize clinical utility. Over 50% of Americans use some form of integrative and functional medicine with 20% using nutritional supplements or specific botanical formulations to complement their health care needs . ConcernAnanas comosus (L.) Merr. and is widely utilized as an anti-inflammatory compound due to its endogenous protease activity [http://merops.sanger.ac.uk/) and may refer to a mixture of cysteine proteinases, peroxidases, acid phosphatases, glycosidases, and inhibitory proteins found throughout the pineapple fruit (EC 3.4.22.33) and stem (EC 3.4.22.32) [Stem bromelain (Br) is an extract of the common pineapple activity . Br is c4.22.32) , 20. His4.22.32) –23. Alth4.22.32) –29. in vivo biomarkers for this botanical drug [The degree to which Br and its components are absorbed and retain function still remains to be fully elucidated. In experimental models of irritable bowel disease, it has been reported that the majority of orally administered Br remains undegraded and retains its enzymatic activity in the intestinal lumen of mice . When adcal drug –37. The purpose of this study was to validate the components of a clinically used stem Br product, using MS and to determine if a Br peptide biomarker could be identified in the plasma of Br treated mice using LC-MS/MS. This is one of the first studies to report the components within a clinically used Br product and to identify a Br peptide (DYGAVNEVK) in plasma. This approach can now be applied to improve the characterization and quality control of Br in the marketplace. This biomarker will provide an important tool for standardizing the effective dose of the product in order to correlate the absorbed dose with therapeutic effects attributed to this complex product. Ananas comosus Merr. (common pineapple), was confirmed by matching its profile with the industry (Sigma) standards (via HPLC) as previously described [Br  was obtained from RHG & Company Inc., Vital Nutrients  and stored at 4°C in opaque containers. The identity of Br, extracted from escribed . Br was ad libitum. All mouse procedures were approved by the Animal Care Committee at the University of Connecticut Health Center (protocols 2009-508 and 2010-634).Female C57BL/6J mice, 3 to 6 months of age and weighing 17 to 20 g, were purchased from the Jackson Laboratory  and housed conventionally in plastic cages with corncob bedding. The mouse room was maintained at 22 to 24°C with a daily light/dark cycle (light from 0600 to 1800 hours). Chow and water were supplied ex vivo spiked serum experiments 100 mg Br was suspended in 20 ml diH20; 10 μL naïve mouse serum was spiked with 10 μg Br. For i.p. treatment, 60 mg of Br was suspended in 125 mL of physiologic saline as previously described [ Individual stock solutions of stem Br were prepared. For escribed , 40. Aniμg/mL) at 37°C for 4 h. Formic acid (5%) was added to stop the reaction and the supernatant was used for subsequent analyses. Br was processed with a modified RIPA buffer to concentrate plant associated protein and remove plant associated detritus and 1-dimensional SDS-PAGE separation was performed on the extract plant protein. Br samples were loaded into a 10% reducing mini-SDS-PAGE and separated via electrophoresis for 20 min. The gel lane corresponding to Br was then cut into 13 sections and subjected to proteolytic digestion on a ProGest workstation (Genomic Solutions) via reduction with DTT at 60°C, followed by a cool down to room temperature. Samples were then alkylated with iodoacetamide and digested in trypsin  at a flow rate of 10 μL/min. A 30 min gradient elution was conducted over a 15 cm, 75 μm ID C12 column at 300 nL/min. For identification of proteins in Br the mass spectrometer was operated in data-dependent mode in which the six most abundant ions were selected for MS/MS. The Orbitrap MS scan was performed at 60,000 FWHM resolutions as previously described [ Samples were analyzed using nano-ESI with a ThermoFisher LTQ Orbitrap XL LC/MS/MS. 30 escribed , 42. http://www.matrixscience.com/) with the following parameters: type of search MS/MS ion search, taxonomy-all kingdoms, enzyme-trypsin, fixed modifications-carbamidomethyl (C), variable modifications-oxidation (M), acetyl (N-term), pyro-glu (N-term Q), deamidation , mass values-monoisotopic, protein mass-unrestricted, peptide mass tolerance- ± 10 ppm, fragment mass tolerance- ± 0.5 Da, and max missed cleavages-2. Samples were further analyzed using the Scaffold algorithm (http://www.proteomesoftware.com/) with the DAT files generated by Mascot. Positive identification was determined if a minimum of 5 peptides matched per protein with minimum probabilities of 95% at the protein level and 95% at the corresponding peptide level.MS/MS data were first searched using Mascot .Silver staining was used to detect proteins after electrophoretic separation on polyacrylamide gels. As no prominent protein bands were detectable in area of interest, three gel slices from each lane ranging from 23 to 39 kDa areas were excised and in-gel trypsin digestion was performed as described previously . Samples were then resuspended in buffer A  acid in water) and stored at −20°C until further analysis.μm capillary column packed in-house  by means of a micro-autosampler . The column was previously equilibrated with solvent A . Peptides were eluted with a linear gradient from 100% solvent A to 80% solvent B  in 85 min at an elution rate of 200 nL/min. A Hewlett Packard 1100 solvent delivery system with flow splitting  was used. Peptides were eluted directly into the LTQ ESI ion trap mass spectrometer equipped with data-dependent acquisition, and a scan was performed. Each full MS scan was followed by one MS/MS scan of the most intense peaks in the full MS spectrum with dynamic exclusion enabled to allow detection of low abundant peptide ions. Auto sampler loading, mass spectrometric scan functions, and HPLC solvent gradients were controlled by the Xcalibur data system .The peptide digests were sequenced using a high throughput tandem mass spectrometer, LTQ ESI ion trap  equipped with a commercial nanoelectrospray device as described previously . Each sample was directly loaded onto a 10 cm × 100 Ananas comosus) database from the NCBI, National Institutes of Health, and Advanced Biomedical Computing Center using the SEQUEST algorithm (SEQUEST-PVM version 27 (revision 0)) and SEQUEST parameters were as follows: all the filtering thresholds were off; mass tolerance of 1.5 Da for precursor ions and 0.5 Da for fragment ions; full tryptic constraint allowing one missed cleavage; allowing oxidization (+16 Da) of methionine. The database search results were processed using the INTERACT program and filtered with the following criteria: Xcorr cutoff values of 1.8, 1.8, and 3.1 for 1+, 2+, and 3+ peptides, respectively, ΔCn ≥ 0.1. Finally, all potential MS/MS spectral matches were subjected to manual inspection as a final validation step prior to acceptance as a valid peptide identification. The spectral count of identified peptides from three runs of each sample was used for semiquantitative measurement of bromelain-mediated events. All LC-MS/MS runs were processed in the following way as described previously , 44. AllE. coli, Salmonella, S. aureus, and P. aeruginosa and below limits for mold yeast  and Enterobacteriaceae . Aflatoxins  were negative and all heavy metals tested  were below allowable limits. In the present study, the Br raw material was obtained from RHG & Company Inc., Vital Nutrients imported from Thailand. Prior to manufacturing, Br was independently tested for identification and quality. A summary of results is provided in in vivo Br treatment initiated  were critical in standardization of LC-MS/MS for the next phase of experiments, identification, and quantification of a Br peptide in the plasma of mice following Br treatment. Plasma was collected and processed, and the filtrate was analyzed by LC-MS/MS. For the analysis, the most abundant peak in the chromatograph eluded at 5.54 min  for monitoring the absorbed dose or therapeutic effectiveness of the product. Currently manufacturers who produce Br supplements are expected to provide specific details about their ingredients on the label , 47. ThiIn order to characterize Br being utilized by consumers, we chose a commercially available product distributed to physicians for use in our research. Although there were several industrial sources available, most are “for research purposes only” such as Sigma's and may not represent the Br manufactured for medicinal use. Also, handling, testing and consistency of natural products may vary greatly. The Br we utilized was independently tested by analytical laboratories for enzymatic activity and contamination from solvents, microbials, aflatoxins, and heavy metals . OrganolBefore analysis could be applied to the serum of i.p. Br treated mice, the peptides and protein(s) present in Br stem extract had to be identified and verified in order to choose suitable peptide(s) for use as biomarkers. Separation of Br raw material via LC and identification of Br peptides via MS/MS resulted in the identification of five protease : (1) FB1Ananas comosus. Similar jacalin-like lectins have been identified in several plant families  and function in protein-carbohydrate recognition through cell surface binding of carbohydrates such as mannose [In contrast to stem Br, which has been the subject of numerous basic and clinical studies, there are very few studies on individual Br proteins. A novel ananain comosain combination product, Vianain, has been formulated for topical wound treatment and debridement due to its inherent protease activity , and the mannose . Jacalin mannose , 62. 125I-labeled Br was absorbed and present in the serum of rats [Although controversy exists regarding the extent to which Br is absorbed and present in the serum, Castel has identified the presence of anti-Br antibodies in human plasma after oral administration  while in of rats , 64. How of rats . As part|2463588 protein were previously observed in an LC-MS/MS analysis of bromelain extract. When plasma from Br treated mice was evaluated, we identified the DYGAVNEVK biomarker peptide. The total number of spectra identified or spectral count for DYGAVNEVK was found to peak at 6 hrs over the 24 hr time course evaluated after initial intraperitoneal Br treatment. Although the specific activity of this peptide is unknown, DYGAVNEVK is present in one of the four proteolytically active proteins (FB1035) within Br which contribute to its therapeutic effects. Future studies may determine the contribution of individual peptides and set the stage for their use as compounds to standardize Br beyond its peptidase activity, which is becoming the gold standard within the botanical extract industry [The unique peptides DYGAVNEVK, TGYLVSLSEQEVLDCAVSYGCK, and NSWGSSWGEGGYVR within the FB1035 giindustry –68. By u


Cachexia is a state of involuntary weight loss common to many chronic diseases. Experimental data, showing that cachexia is related to the enhancement of acute phase response reaction, led to the new definition of cachexia that included, aside from the principal criterion of weight loss, other “minor criteria”, Amongst them are levels of C-reactive protein (CRP), albumin and hemoglobin. However, there is paucity of data regarding possible differences of these laboratory parameters in patients with various diseases known to be related to cachexia.CRP, albumin and hemoglobin were evaluated in 119 patients, divided into two disease groups, hematological (ones with diagnosis of non-Hodgkin lymphoma or Hodgkin disease) and non-hematological (solid tumor patients and patients with chronic heart failure). Patients were further subdivided into two nutritional groups, cachectic and non-cachectic ones according to the principal criterion for cacxehia i.e. loss of body weight.We found that cachectic patients had higher levels of CRP, and lower levels of both hemoglobin and albumin compared to non-cachectic patients, regardless of the disease group they fitted. On the other hand, the group of hematological patients had lower levels of CRP primarily due to the differences found in the non-cachectic group. Higher levels of albumin were also found in the hematological group regardless of the nutritional group they fitted. Limitations of cut-off values, proposed by definition, were found, mostly regarding their relatively low sensitivity and low negative predictive value.As expected, differences in values of routine laboratory parameters used in definition of cachexia were found between cachectic and non-cachectic patients. Their values differed between hematological and non-hematological patients both in cachectic and non-cachectic group. Cut-off levels currently used in definition of cachexia have limitations and should be further evaluated. Cachexia is a state of involuntary weight loss common to many patients with chronic diseases such as cancer, acquired immunodeficiency syndrome, congestive heart failure, chronic infections, rheumatoid arthritis and kidney failure ,2. It waThe mechanism of development of cachexia in all these conditions is believed to be similar if not the same . CachexiRelation between chronic inflammation, as a trigger for development of cachexia, and the subsequent changes in certain clinical and laboratory features lead key opinion leaders to incorporate, beside principal criterion for the diagnosis of cachexia which is at least 5% loss of edema – free body weight during the previous 12 months or less, other “minor criteria” into the new definition of cachexia. Amongst those “minor criteria” are elevated levels of CRP (≥ 5 mg/l) and decreased levels of hemoglobin (≤ 12 g/dl) and albumin (≤ 3.2 g/dl)  which weAlthough it is well known that cachexia occurs in different diseases, comparative clinical or laboratory studies are lacking. Aim of the present study was to investigate whether there are differences between aforementioned routine laboratory parameters between patients with hematological diseases and other patients with diseases known to be related to cachexia . First, we examined those parameters in all patients included in this study, thereafter investigating same parameters in cachectic and non-cachectic patients. Finally, we examined the proposed cut-off values of CRP, hemoglobin and albumin for diagnosis of cachexia, in order to establish their clinical usefulness.The study was conducted at the Department of Internal Medicine of University Hospital Merkur in Zagreb. Patients' data were collected from May 2011 until February 2012 on consecutive patients that were eligible for entry. A diagnosis of either solid hematological tumors , other solid tumors of any site or chronic heart failure of any etiology was mandatory. Diagnosis of malignant disease had to be proven with adequate histopathological sample. Diagnosis of heart failure was made according to the guidelines criteria .Exclusion criteria were age of less than 18 years, starvation, malabsorption, diarrhea, active thyroid disease, depression or other severe psychiatric disease, chronic obstructive pulmonary disease, renal insufficiency of grade ≥ 3, myocardial infarction in less than last 12 weeks, liver insufficiency, neuromuscular diseases, alcohol or drug abuse and previous use of cardiotoxic chemotherapy in doses proven to be cardiotoxic .The study received previous local ethical board approval and was conducted according to the Declaration of Helsinki and its subsequent amendments. All patients gave written informed consent before participation in the study.Complete history was recorded, full physical examinations were performed and blood samples were collected at baseline. Baseline blood analyses included complete blood count and complete biochemistry . All measurements were done according to the standard protocol of institutional laboratory.Patients were considered to be cachectic if they fulfilled main criteria for the diagnosis according to the consensus document. This criterion is loss of ≥ 5% of body weight in last 12 months or less (no less than 6 months). Other criteria , of which 3 of 5 need to be fulfilled for diagnosis, were also sought but we did not include them in the stratification of our patients .Patients were further divided into two disease groups, hematological (ones with diagnosis of non-Hodgkin lymphoma or Hodgkin disease) and non-hematological (solid tumor patients and patients with chronic heart failure).Measurements of CRP and albumin were performed on the Beckman Coulter Olympus AU 680 analyzer  using Beckman Coulter System reagents and standardized laboratory methods on fresh sera on the day of blood collection. Serum was collected after centrifugation for 15 minutes at 3000 rpm. CRP concentrations were determined by automated high-sensitivity latex-enhanced immuno-turbidimetric assay. The calibrator CRP values were traceable to CRM 470. The limit of detection was 0.1 mg/L. Quantitative determination of albumin in serum was performed using Beckman Coulter bromocresol green photometric color test. Measurements of hemoglobin was performed on the Sysmex XE-2100 , fully automated discrete hematology analyzer designed to generate complete blood counts. Hemoglobin determination was achieved using the sodium laurel sulfate (SLS)-hemoglobin method. Participation of Institute of Clinical Chemistry and Laboratory Medicine University Hospital Merkur accredited according to ISO 15189  in the International External Quality Assessment Schemes for general and special medical biochemistry organized by World Health Organization for Laboratory Hematology (IEQAS - H) and by LABQUALITY, Finland for biochemical tests, confirms clinical reliability of obtained laboratory test results .The results were expressed as the mean +/− standard deviation or as a proportion of the total number. Analyses of specificity, sensitivity, positive predictive value and negative predictive value were done using appropriate formulas. Differences in proportions  were compared using chi square test. Mann Whitney test was used to test the equality of continuous variables. A p-value < 0.05 was considered statistically significant. All statistics were performed with the StatView™ statistical program, version 5.0.1. .One hundred nineteen  consecutive patients were enrolled. Fifty five patients were from the hematological group (45 had diagnosis of non-Hodgkin lymphoma and 10 had diagnosis of Hodgkin disease). In the non-hematological group 43 patients were diagnosed with solid tumor of various sites  and 21 had chronic heart failure (15 had ischemic cardiomyopathy and 6 had non-ischemic cardiomyopathy). In the group of heart failure patients 2 (10%) were in NYHA class I, 8 (38%) patients were in stage II, 9 (42%) were in stage III, and the remaining 2 (10%) in stage IV.Hematological patients were significantly younger than non hematological patients. We found no statistically significant differences in prevalence of cachexia, lost body weight in cachectic patients or period of loss of body weight in cachectic patients between prespecified disease groups. Patient clinical characteristics are shown in Table Mean value of CRP in the whole group was 22.78 mg/l (+/−37.5). Hematological patients had mean value of CRP 15.98 mg/l (+/−36.1) and non-hematological patients had a mean value of CRP 28.71 mg/l (+/−39.1). Observed difference was found to be statistically significant (p = 0.048). When we subdivided patients according to their nutritional status, cachectic patients as a whole group had a mean CRP value of 34.18 mg/l (+/−51.9) and patients without cachexia had a mean value of CRP 17.57 mg/l (+/−27.5) which was also significantly different (p = 0.021). On the other hand, we could not find significant differences in CRP levels in hematological (32.90 mg/l (+/−64.5) vs. 9.75 mg/l (+/−12.9); p = 0.1) or in non-hematological (35.26 mg/l (+/−43.0) vs. 25.55 mg/l (+/−35.6); p=0.27) group when we divided them according to their cachectic status. In fact the observed initial difference was related to the difference between hematological and non-hematological patients in the non-cachectic group (9.75 mg/l (+/−12.9) vs. 25.55 mg/l (+/−35.6); p = 0.01). CRP levels according to the predefined groups are shown in Table Mean value of hemoglobin in all examined patients was 12.53 g/dl (+/−2.1). Cachectic patients had lower levels of hemoglobin (11.73 g/dl (+/−2.0) vs. 12.90 g/dl (7.9-16.2); p = 0.039) than non-cachectic patients. There was no difference in values of hemoglobin between hematological and non-hematological patients (12.70 g/dl (+/−2.3) vs. 12.38 g/dl (+/−1.9); p = 0.33). All other analyzes done in the same way as for CRP values showed only statistically significant difference in hematological group depending on their nutritional status (11.20 g/dl (+/−2.2) vs. 13.27 g/dl (+/−2.0); p = 0.003). Hemoglobin levels according to the groups are shown in Table Mean level of albumin for all patients was 38.3 g/l (+/−6.5). Cachectic patients had lower levels of albumin (34.95 g/l (+/− 6.5) vs. 39.85 g/l (+/−5.8); p = 0.0002) both as a group and when divided into hematological (38.05 g/l (+/−6.1) vs. 42.04 g/l (+/−5.2); p = 0.019) and non-hematological ones (32.83 g/l (+/−6.1) vs. 37.85 g/l (+/−5.7); p = 0.004). On the other hand, hematological patients had higher level of albumin than non-hematological patients (40.95 g/l (+/−6.1) vs. 36.01 (+/−6.2); p = 0.0001) regardless of their nutritional status. Values of albumin levels are shown in Table Finally, we analyzed values of proposed cut-offs for CRP (≥ 5 mg/l), hemoglobin (≤ 12.0 g/dl) and albumin (≤ 32.0 g/l) both separately and together in the whole group of patients and upon dividing them into hematological and non-hematological groups. Sensitivities for the whole group were found to be 76% for CRP, 59% for hemoglobin, 35% for albumin and 86% for either of them being positive. Specificity was 39% for CRP, 68% for hemoglobin, 85% for albumin and 29% for either of them being positive. Negative predictive values were 78% for CRP, 79% for hemoglobin, 74% for albumin and 83% for all of them being negative. On the other hand, positive predictive value for CRP cut-off was 36%, for hemoglobin 46%, for albumin 52% and for either of them being positive 35%.In hematological group, sensitivity of CRP was 73%, specificity 50%, negative predictive value was 83% and positive predictive value 35%. For hemoglobin, values were 73%, 75%, 88% and 52% and for albumin they were 25%, 92%, 75% and 50%, respectively. For all three values being either above or below proposed cut-offs values were 87%, 37%, 88%, 65%. Similar data were observed in non-hematological group and were for CRP for sensitivity 77%, 29% for specificity, 70% for negative predictive value and 36% for positive predictive value. For hemoglobin values were 50%, 62%, 70% and 40% and for albumin they were 45%, 79%, 73% and 53%. For all three cut-offs we got 92% sensitivity, 21% specificity, 75% negative predictive value and 45% of positive predictive value. Data for CRP, hemoglobin and albumin are shown in Figures Proposed cut-off for CRP did not significantly differentiate cachectic and non-cachectic patients neither in a whole group (p = 0.12) nor in hematological (p = 0.12) or non-hematological (p = 0.61) patients separately. Hemoglobin cut-off was able to discriminate cachectic and non-cachectic patients in the whole group (p = 0.043) and in hematological patients (p = 0.01) but not in non-hematological group of patients (p = 0.36). On the other hand, albumin cut-off discriminated well between cachectic and non-cachectic patients both in all patients (p = 0.011) and in non-hematological patients (p = 0.04), but not in hematological patients (p = 0.18). All three values being above or below the proposed cut-offs could not discriminate between cachectic and non-cachectic patients observed either together (p = 0.06) or separately as hematological (p = 0.08) or non-hematological (p = 0.44).Before discussing further our results we believe that three possible questions, that may arise reading this paper, need to be clarified. Firstly, the rational to divide our patients into hematological and non-hematological group. Would it not have been more appropriate to classify them into those with tumor disease  and those with chronic benign state (chronic heart failure)? Would it not have been more appropriate to compare patients in the three separate groups i.e. hematological, solid tumors and heart failure group? There are several explanations for our decision. Our institution is a referral center for hematological diseases and most of the patients admitted to our institution indeed suffer from hematological diseases. Fewer patients are admitted for solid tumors, heart failure and other diseases related to cachexia. By dividing our patients in hematological and non-hematological ones we have achieved optimal balance of consecutive patients in respective groups. However, the answer is not just of technical or statistical matter. If one would be really interested, and it was indeed our intention, to investigate differences between hematological and patients with other diseases related to cachexia how is one supposed to divide patients? Dividing patients into three groups, apart from creating numerically unbalanced groups would not answer this question. In fact, in order to get the correct answer to this question a comparison between hematological patients and all other groups of patients with diseases that can cause cachexia would be necessary. Unfortunately, such a study could not have been conducted in our institution although we eagerly await a study by others that would address this question.Secondly, we decided to divide patients into cachectic and non-cachectic just according to the main criterion that is loss of at least 5% of body weight during the previous 12 months or less, not further looking at other criteria proposed by new definition that we refer to . We thin… .time frame may be disease specific and is likely to be shorter in cancer (3–6 months) and longer in chronic heart failure and kidney disease (12 months)… . .”. Although this sentence may imply the upper limit of 12 months we believe that it also may imply periods longer than 12 months. Some of the published data refers to periods exceeding 12 months, as was the case in one of the pivotal studies from Anker et al. with duration of weight loss at inclusion up to 13 months. After inclusion patients were followed for a mean of 686 days which is almost two years [And lastly we decided, as is obvious from our patient characteristics, to enroll patients that had weight loss for more than 12 months. We think that it is justifiable, although the definition that we refer to  states two years . In thatGiven the scarcity of data regarding clinical and laboratory features comparing patients with various diseases that are related to cachexia we conducted our study to address some of these issues. We were specifically focused on differences between hematological and non-hematological patients. We found, as others did, that cachectic patients as a group, had significantly higher levels of CRP and lower levels of both albumin and hemoglobin. Secondly, we divided patients into hematological and non-hematological groups and found that hematological patients had significantly lower levels of CRP and higher levels of albumin. There was no difference in levels of hemoglobin. When we further subdivided patients into cachectic and non-cachectic groups we found that the differences observed for CRP were mainly due to differences found in non-cachectic group. Observed differences in albumin levels were present regardless of this subdivision. Hematological cachectic patients had lower levels of hemoglobin than non-cachectic ones. The same was not true for non-hematological patients. Observed differences could imply possible divergences in pathophysiology between the groups of patients as we divided them. Unfortunately, we were not able to conduct investigations regarding possible differences in for example expression of inflammatory cytokines that could explain observed differences. Because of that our question “is there only one cachexia?”, although provocative, remains purely speculative and awaits further trials with larger numbers of patients and more balanced groups exploring other features of cachexia, probably primarily those incorporated into proposed definition . New proWe then evaluated proposed cut-offs for CRP (≥ 5mg/L), hemoglobin (≤ 12 g/dl) and albumin (≤ 3.2 g/dl) and found that CRP had highest sensitivity, followed by hemoglobin and albumin. Inverse relationship was found for specificity. Cut-offs were found to have relatively good negative predictive value whereas positive predictive value was poor. Combining all three cut-offs just emphasized above mentioned relations. When we divided patients to the hematological and non-hematological groups, proposed cut-offs followed same trends as in the whole group. Proposed cut-off for CRP was not able to distinct cachectic from non-cachectic patients in the whole group nor was it able to distinct them when we further subdivided them into hematological and non-hematological groups. On the other hand, proposed cut-off for albumin was able to distinct patients according to their nutritional status regardless of further subdivisions. Hemoglobin cut-off showed its predictive power in both whole group as well as in hematological group but not in non-hematological group. Although such results would imply that CRP cut-off is least effective and albumin cut-off is the most effective one such conclusions would need larger studies powered enough even to give more precise values. Until then, our results serve as ones that arouse suspicion in proposed cut-offs.Patients suffering from diseases related to cachexia have elevated values of routine laboratory parameters related to enhanced inflammation. We found significant differences between these values for cachectic and non-cachectic groups of patients. We also revealed some significant differences between these values when dividing patients into hematological or non-hematological groups, although we are well aware of the relatively small number of patients in groups and of the variability of patients’ diagnoses especially in the group of patients with solid tumors. We are also aware of the difference in age between the hematological and non-hematological group that could have influenced our results. These restrictions stopped us from attributing observed differences to differences in pathophysiological mechanisms of exaggerated immune response in different diseases. On the other hand, limitations of our study certainly cannot allow anyone to conclude that those differences in fact actually do not exist. To our knowledge, this is the first study that tries to compare between groups of patients suffering from the different diseases that can cause cachexia. Further studies with larger numbers of patients and with smaller variability inside the groups are warranted for further clarification of possible differences between the cachexia presenting in various diseases.CRP: C- reactive protein; H: Hematological; NH: Non-hematological; C: Cachectic; NC: Non-cachectic.The author’s declare that they have no competing interests.TL gathered all the patients’ data and blood samples and participated in the design of the study and statistical analyses. SP and ZFM conducted laboratory analyses vital to the study. RV participated in the design of the study and statistical analyses. All authors read and approved the final manuscript.