Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes. Analogues of microbial secondary metabolites, which include many antibiotics and antitumor drugs, can be engineered from unusual primer units of the polyketide backbone to create new medicinal compounds with promising novel pharmacological properties Polyketides are a large class of structurally and pharmacologically diverse molecules, including many antibiotics and antitumor drugs . They armodule used in this report refers to a collection of dissociated enzymes. The elongation module consists of enzymes involved in chain extension steps of polyketide biosynthesis, while the initiation module consists of enzymes involved in the nonacetate priming of certain aromatic PKSs.) However, the polyketide backbones of most bacterial aromatic polyketides (act) biosynthetic pathway synthesizes an octaketide (C16) backbone from eight malonyl-CoA equivalents, whereas the tetracenomycin (tcm) minimal PKS synthesizes a decaketide (C20) backbone from ten equivalents of malonyl-CoA , zhuH (KS), and zhuG (ACP) genes from the R1128 gene cluster were coexpressed with the genes encoding the act KS-CLF, ZhuN (ACP), and the act KR (on plasmid pYT46). Control plasmids pYT44, lacking the R1128 initiation module, and pYT45, lacking zhuN, were also constructed and characterized. All three plasmids were introduced into S. coelicolor CH999 by transformation, and polyketides products were analyzed by liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) spectroscopy.Guided by our recent observation that the KS–ACP pairs of initiation and elongation PKS modules have orthogonal molecular recognition features, we first attempted to coexpress the R1128 initiation module with the S. coelicolor CH999/pYT44 produced mutactin , the expected products of the act minimal PKS in the presence of act KR (1) and YT46b (2), had structures shown in act KR. Supplementing the growth medium with13C-labeled sodium propionate revealed that the alkyl moiety of 1 was derived from propionyl-CoA. Similarly, supplementing the growth medium with 1 g/l of L-valine resulted in a 2-fold increase in the level of 2, suggesting the branched alkyl group observed in 2 was derived from isobutyryl-CoA, a primary catabolite of L-valine f act KR , whereasf act KR A , has a remarkably strong preference for the diketide product of the R1128 initiation module. However, unexpectedly, the incorporation of a longer chain substrate into the catalytic cycle of the act minimal PKS results in a reduced number of malonyl units utilized during iterative chain extension  an initiation module; (ii) a decaketide minimal PKS that can extend the five to six carbon products of the initiation module by seven more extender units to yield a 19–20 carbon polyketide; and (iii) appropriate ARO and CYC subunits.To validate the generality of the above observations, we sought to reconstitute the biosynthesis of R1128 family of compounds in  modules . It follact ARO/CYC family are unable to cyclize an unreduced octaketide  . Thus, ZhuI and ZhuJ are able to cyclize an unreduced octaketide into the corresponding anthraquinone. The incomplete transformation of nascent octaketides into TMAC may be due to the fact that an acetate-primed octaketide is not a natural substrate of the two CYCs (see below).The R1128 family of antibiotics represents a unique set of anthraquinones that contain an unreduced C-9 carbonyl (present as an enolic C-9 hydroxyl in R1128). Since members of the taketide  and sinclization , AROs anlization . ZhuI, wYT92 see  were concompound , 3,6,8-ttcm KS-CLF, ZhuN, ZhuI, ZhuJ, and the R1128 initiation module. Plasmid pYT92 (which lacks the initiation module) was used as the negative control. In addition to the decaketides SEK15 and SEK15b, two new anthraquinone compounds, YT128 (3) and YT128b (4), were isolated at comparable levels (7 mg/l each) from S. coelicolor CH999/pYT128. The two compounds account for 50% of total polyketides produced by this recombinant strain. [13C]Propionate and valine feeding experiments verified the alkyl groups installed at C-16 were indeed derived from either propionyl-CoA or isobutyryl-CoA. NMR and MS analyses confirmed the identities of 3 and 4 as alkyl-primed TMAC analogs (5) and R1128c (6) , were present at an approximately 20% level to that of 3 and 4.The identification of ZhuI and ZhuJ as the appropriate CYCs for the synthesis of R1128-like anthraquinones prompted the design of pYT128, which coexpresses  analogs . The nat3–6 further validated our hypothesis that the initiation module of the R1128 PKS could productively interact with elongation modules from any bacterial aromatic PKS. Moreover, by incorporating a primer unit with at least five carbon atoms, the tcm KS-CLF effectively became an octaketide synthase with respect to the rest of the polyketide molecule. This was analogous to the conversion of the act KS-CLF into a hexaketide synthase in the presence of the R1128 initiation module. It also suggested that the R1128 KS-CLF was intrinsically a decaketide synthase. Finally, analogous to the act KR, ZhuI and ZhuJ were programmed to recognize full-length polyketide chains based upon the number of β-carbonyl groups, rather than the carbon chain length of the backbone. Our findings below suggest this is a general property for all CYCs. It should be noted that the CYCs ZhuI and ZhuJ were apparently more efficient in processing the unreduced alkyl-primed octaketide than an acetate-primed octaketide, since no alkyl-primed analogs of SEK4 and SEK4b were observed in extracts of CH999/pYT128. .The engineered biosynthesis of compounds 8 and 9  lacking the R1128 initiation module was also constructed, transformed, and analyzed.To demonstrate the utility of hybrid bimodular PKSs for the rational design of new analogs of known polyketides, we targeted the engineered biosynthesis of alkyl-primed 3,8-dihydroxy-methylanthraquinone carboxylic acid (DMAC) analogs 8 and 9 . Specifitcm minimal PKS outfitted with the act KR produced the expected polyketides RM20, RM20b, and RM20c  in CH999/pYT127. The identities of 8 and 9 were verified by NMR and MS  and 6   , suggest50 = 5 nM) of the glucose-6-phosphate translocase enzyme complex, an attractive target for the treatment of Type II diabetes , DMAC, 3, 4, 7, 8, or 9 using a colorimetric assay described earlier  a long substituent at C-16 in an anthraquinone is important for targeting the membrane-bound G6Pase, and (ii) the C-9 position of the anthraquinone can be chemically modified without significantly affecting the enzyme-inhibitor interactions.The integrity of the microsomes was first verified by comparing the glucose-6-phosphatase (G6Pase) activity in the presence of either glucose-6-phosphate or mannose-6-phosphate. The activity of G6Pase was then measured in the presence of chlorogenic acid , the former class of products may not be derived via decarboxylative priming in strains carrying bimodular PKSs. Instead, they may arise as a result of premature diketide transfer from the initiation module to the elongation module before the β-carbonyl can be reduced. Future isotope labeling studies on such systems should be useful for quantifying the distribution between polyketide chains derived from bimodular PKSs versus those that arise via decarboxylative priming.Most bacterial aromatic PKSs catalyze chain initiation by decarboxylating an ACP-bound malonyl extender unit to yield an acetyl primer unit, a reaction that is catalyzed by the KS-CLF. In order to install nonacetate primer units in an aromatic polyketide backbone, one must bypass this decarboxylative priming mechanism. Genetic and enzymological analysis of the R1128 PKS, which utilizes a range of nonacetate primer units, has revealed the existence of two PKS modules. Each module includes a distinct KS and an ACP. Previous studies have shown that these two KS-ACP pairs have orthogonal molecular recognition features, leading to the speculation that the initiation module may be able to productively interact with other bacterial aromatic PKSs to synthesize hybrid polyketides. However, the ability of the R1128 initiation module to kinetically compete with the intrinsic decarboxylative priming mechanism of the heterologous PKS was unexplored. To address this question, we coexpressed the entire R1128 initiation module with either the Streptomyces roseofulvus can synthesize both nanaomycin (an acetate-primed aromatic polyketide) and frenolicin   . They al analog) . Thus, nact, frn, tcm, and whiE (16–C24 backbones) to yield a range of reactive backbones whose subsequent fates can be controlled by previously analyzed auxiliary PKS subunits and tailoring enzymes.The potential for recombining naturally occurring initiation and elongation PKS modules from bacterial aromatic PKSs is enormous. Other than the R1128 biosynthetic pathway, initiation modules with attractive primer unit specificity can also be found in the doxorubicin, frenolicin, enterocin, and (presumably) oxytetracycline biosynthetic pathways. It should be possible to recombine these synthase units with elongation modules from the and whiE  PKSs  primer units.Our results demonstrate that, since chain length specificity of KS-CLF heterodimers is primarily dictated by the backbone size, incorporation of bulky, nonacetate primer units is compensated for by a reduced number of condensation cycles. Thus, hexaketides and octaketides are synthesized by the act KR selectively reduced the C-9 keto group in an acetate-primed octaketide (DMAC), an alkyl-hexaketide (see act ARO and CYC process a reduced (but not unreduced) octaketide, regardless of the primer unit, although they are unable to recognize hexaketides or decaketides. In contrast, the R1128 CYCs act upon an unreduced, but not reduced, backbone, regardless of the primer unit.In contrast to KS-CLF subunits, the regioselectivity of auxiliary PKS enzymes such as KR, ARO, and CYC is unaffected by the incorporation of nonacetate starter units. For example, the tide see B, and antide see B. This otide see  that succatk, approximately150 s–1)  , whereasfrn PKS, are occupied by bulkier residues, thus excluding acyl groups larger than acetyl-CoA. Therefore, altering the size and polarity of these gatekeeping residues using rational mutagenesis and directed evolution may enlarge the repertoire of acyl-CoA moieties recognized by KSIII enzymes.To further expand the repertoire of primer units that can be introduced into aromatic polyketides using bimodular PKSs, one could (i) alter the substrate specificity of the R1128 initiation module and/or (ii) engineer in vivo metabolic pathways for new types of primer units. KSIII homologs found in initiation modules serve as gatekeepers in primer unit selection and are therefore attractive targets for protein engineering. The X-ray crystal structure of ZhuH has recently been solved and has led to the identification of a binding pocket for the acyl-CoA moiety . ZhuH adS. coelicolor is the primary source of primer units such as isobutyryl-CoA  and isovaleryl-CoA (leucine). These pathways involve transamination  to convert the amino acid into the corresponding α-ketoacid, followed by decarboxylation  to yield the corresponding acyl-CoA . PCR products were first cloned into pCRBlunt vector , followed by DNA sequencing . Unmethylated DNA was obtained by using the methylase-deficient strain GM2163 .zhuC: 5′-CCTCTAGATGTACTCGGGTCGAGGAGACCTCCG-3′, 5′-GGACTAGTGCCACGTTCACCGTTCCGCCGCG-3′; zhuN: 5′-CATGCGACCCGTCTAGAGAAGGAGATTCCG-3′, 5′-CGCGGTTCTGCACTAGTCAGGCCGCGGCC-3′; zhuG: 5′-CCTGTCTAGAGGGAGGACGAACCC-3′, 5′-TGCTGCAGTCAGCCCGCGGTCTCG-3′; zhuH: 5′-GACTGCAGCAGAACCGCGAAAGGTGG-3′, 5′-AGTAGTACGTTTAAACTCAAGCCGGAGTGGACGGC-3′; actVII/actIV: 5′-GCCGTTTAAACGCTGGCGCCAAGCTTCTC-3′, 5′-CCGGAGACGCGTCACGGCCGAAGC-3′; zhuI/zhuJ: 5′-GCCGTTTAAACCGAGGAGCACCCTCATGCGTC-3′, 5′-GGACTAGTCCTCCTCTTCCTGCTCG-3′. The introduced restriction sites are shown in italics. Genes encoding zhuC, zhuH, zhuN, zhuG, and zhuI/zhuJ were amplified from pHU235  and zhuI/zhuJ (1.3 kb) were each cloned as a PmeI–EcoRI cassette. Different combinations of cassettes, as shown in actKS/CLF), pSEK24 (actKS/CLF), pSEK33 (tcmKS-CLF), or pRM20 (KR-tcmKS/CLF) to yield pYT46, pYT105, pYT128, and pYT127, respectively.The following primers were used to amplify the individual genes: m pHU235 , and genrom pRM5 . zhuC, zR, minutes) were as follows: 1: 13.8; 2: 15.4; 3: 20.4; 4: 21.8; 5: 24.9; 6: 26.4; 7: 17.1; 8: 22.8; 9: 24.4; 10: 27.7; 11: 29.4. LC/MS was performed at the Vincent Coates Foundation Mass Spectrometry Laboratory at Stanford University using a ThermoFinnigan  quadrupole ion trap LC/MS system and electrospray ionization (both positive and negative ionization).The strains were grown on R5 plates containing 50 mg/l thiostrepton at 30°C for 7–10 d. Acyl-CoA precursors such as sodium propionate and valine were added at 1 g/l when needed. For LC/MS and analytic HPLC analysis, one plate was sufficient. The plate was chopped into fine pieces and extracted with 30 ml of ethyl acetate (EA)/methanol/acetic acid (89:10:1). The solvent was removed in vacuo and the residue was redissolved in 1 ml of methanol. The polyketide products were separated and detected by analytical reversed-phase HPLC using a diode array detector at 280 and 410 nm using an Alltech  Econosphere C18 column (50 mm × 4.6 mm); linear gradient: 20% acetonitrile (ACN) in water (0.1% TFA) to 60% ACN in water (0.1% TFA) over 30 min; 1 ml/min. HPLC retention times (t1 (Rf = 0.34) and 2 (Rf = 0.41) were developed with EA/methanol/acetic acid (97:2:1), while those spotted with 3 (Rf = 0.29), 4 (Rf = 0.37), 7 (Rf = 0.21), 8 (Rf = 0.34), and 9 (Rf = 0.43) were developed with EA/hexane/acetic acid (90:10:1). The desired bands were excised from the TLC plates and stirred in EA/methanol  for 2 h. The compounds were eluted from silica using the same solvent and dried in vacuo.Sufficient number of R5 plates  streaked with the desired CH999 strains were grown at 30°C for 1 wk. The plates were chopped into fine pieces and extracted with a minimum of 1 l of EA/methanol/acetic acid (89:10:1). The organic solvents were removed and the residuals were dissolved in 5 ml of methanol. The solution was filtered and injected into a preparative reversed-phase HPLC column . A 20%–60% ACN in water (0.1 % TFA) gradient  was used to separate the polyketide products. Fractions containing the desired polyketides were combined and concentrated in vacuo. The residuals were redissolved in acetone and applied to a preparative TLC plate . TLC plates spotted with 1H and 13C NMR spectra data are shown in 1: 277.1082 ; 2: 291.1247 ; 3: 355.0823 ; 4: 369.0981 ; 7: 313.0354 ; 8: 339.0871 ; 9: 353.1036 .NMR spectra were recorded on Varian  Inova 500 or Mercury 400 instruments and calibrated using residual undeuterated solvent as an internal reference.50 values were measured after 5 d.The studies were performed as described by Hori et al (1993). The cells were maintained at 37°C and growth was measured with the colorimetric MTT assay after each day. The ICTM . Aliquots  in 0.25 M sucrose were stored at –80°C. Compound stock solutions were prepared in 95% ethanol and diluted with DMSO. Glucose-6-phosphate and mannose-6-phosphate were purchased from Sigma .Male rat liver microsome (Sprague Dawley) was purchased from BD Gentest2, and 1 mM DTT at pH 7.2), 3 μl of glucose-6-phosphate , and 3 μl of inhibitor sample in DMSO. The reaction mixture was incubated at room temperature, and 13 μl of reaction mixture was taken every 10 min and quenched with 117 μl of working solution . The blue reduced phosphomolybdate complex is formed after incubation at 50°C for 20 min. The absorbance was measured at 820 nm.The integrity of microsomes and G6Pase activity was measured based on the colorimetric reaction of inorganic phosphate as previously reported . The enzwww.ebi.ac.uk/swissprot/) accession numbers for the proteins and genes discussed in this paper are act ARO/CYC (Q02055), act KS-CLF (Q02059 and Q02062), ZhuA (Q9F6E1), ZhuB (Q9F6E0), ZhuC (Q9F6D6), ZhuG (Q9F6D5), ZhuH (Q9F6D4), ZhuI (Q9F6D3), ZhuJ (Q9F6D2), and ZhuN (Q9F6C8).The SwissProt (


Metallopanstimulin (MPS-1) is a ribosomal protein that is found in elevated amounts in the sera of patients with head and neck squamous cell carcinoma (HNSCC). We used a test, denoted MPS-H, which detects MPS-1 and MPS-1-like proteins, to determine the relationship between MPS-H serum levels and clinical status of patients with, or at risk for, HNSCC.A total of 125 patients were prospectively enrolled from a university head and neck oncology clinic. Participants included only newly diagnosed HNSCC patients. Two control groups, including 25 non-smokers and 64 smokers, were studied for comparison. A total of 821 serum samples collected over a twenty-four month period were analyzed by the MPS-H radioimmunoassay.HNSCC, non-smokers, and smokers had average MPS-H values of 41.5 ng/mL, 10.2 ng/mL, and 12.8 ng/mL, respectively (p = 0.0001).We conclude that MPS-1 and MPS-1-like proteins are elevated in patients with HNSCC, and that MPS-H appears to be a promising marker of presence of disease and response to treatment in HNSCC patients. Effective therapy for head and neck squamous cell carcinoma (HNSCC), which constitutes approximately 95% of head and neck malignancies, is dependent upon early diagnosis and intervention. Despite the obvious advantage to earlier diagnosis of head and neck malignancies, no strategy has proven to effectively detect these tumors at early stages. Most head and neck neoplasms are detected when the patient has become symptomatic from the effects of the primary disease or when lymphatic metastases are palpable. These tumors are infrequently found incidentally on physical exam, and in these cases are often discovered at an earlier stage. Stage of disease at time of diagnosis is the primary metric used for determination of therapy and prognostication of life expectancy . As tumoSurveillance in the post-treatment head and neck cancer patients has traditionally centered on regular physical examination ,3. OfficMetallopanstimulin-1 (MPS-1) was identified, cloned and characterized in the laboratory of Dr. Fernandez-Pol from a cDNA library constructed from a human mammary carcinoma cell line (MDA-468) that was stimulated by the growth factors TGF-β1 and EGF in the presence of cyclohexamide . MPS-1, Conventionally, ribosomal proteins are thought to be confined in their function to intracellular protein synthesis. Many recent reports have drawn attention to "extraribosomal functions" of ribosomal proteins -20. MoreThe physiology of MPS-1 expression and our initial experience with this protein in HNSCC has led us to conclude that MPS-1 and MPS-1-like proteins may be useful markers in the effort to screen for and analyze the extent of HNSCC . The purA total of 125 volunteers with newly diagnosed HNSCC were prospectively enrolled from a university head and neck oncology clinic. Serum collection consisted of a pre-treatment specimen followed by collections every six weeks during the first year and quarterly during the second year. At the time of specimen collection, presence of HNSCC was judged by all available data including: physical examination (that included an office endoscopy when indicated), biopsy, and radiology . A clinical assessment was rendered based on all available data as "no evidence of disease (NED)", "alive with disease", or an indeterminate status. Serum MPS-H levels in these patients (N = 709) were compared to two control groups. The first control group was comprised of 25 normal, healthy, non-smoking volunteers. The second control group included 64 actively smoking volunteers who were screened for HNSCC and were found to be free of disease during the 1999 Yul Brynner Head and Neck Screening day in St. Louis, Missouri. A total of 821 serum samples collected over a twenty-four month period were analyzed by the MPS-H radioimmuno assay . All patients gave informed consent under an IRB-approved protocol.Technical details for the preparation of reagents for MPS-H antigen determinations, RIA procedure, and patient sample preparation are published elsewhere ,10. Each). Statistical significance was defined as p < 0.05.Average MPS-H levels were compared between HNSCC patients and the two control groups and across American Joint Committee on Cancer (AJCC) stages and subsites using analysis of variance (ANOVA). Based on the results from the clinical assessment we computed the receiver operating characteristic (ROC) curve for MPS-H. The ROC curve plots the trade-off between sensitivity and specificity for a range of threshold values for defining a positive result. All ANOVA and contingency table analysis were performed using SAS  statistical software. ROC curve analysis was performed using R software, an open source implementation of the S language  curve for the MPS-H levels. Figure 4N0M0 SCC of the floor of mouth with positive tumor margins on the cut edge of the mandible, having failed a recent limited surgery by another surgeon. Her presenting MPS-H value was borderline positive when she had little clinical disease [1 on the x axis] and rose immediately postoperatively. A transient rise after surgical ablation or induction chemotherapy is a documented phenomenon observed with numerous tumor markers . The elevation likely results from a large initial disruption of cells within the tumor resulting in a dumping of intracellular MPS-1 into the circulation. This usually returns to baseline in 4 to 6 weeks if the tumor is successfully treated. The patient was clinically NED in the immediate postoperative period and during radiation treatment. Approximately 6 months post operatively, the patient developed a neck mass and by the next visit skin metastasis were seen. On last follow-up, the patient had progression of skin and neck metastasis at which time she decided to pursue hospice care. She expired 6 weeks later.We have observed several instances where elevated MPS-H levels in patients presenting with head and neck neoplasms dropped to normal levels following successful therapy. We have also noted examples of persistent elevations or increases in MPS-H levels in patients with failure to respond to therapy or with recurrence of tumor respectively. Several cases of patients presented in Figures 4N2cM0 SCC of the larynx, which required total laryngectomy, bilateral neck dissections, and postoperative radiation therapy. The patient joined this study 3 months following his surgery, while clinically NED, and has remained free of clinical recurrence for 24 months. The patient had three FDG-PET scans at six-month intervals following his surgery that were all negative for recurrent disease or metastasis. His persistently low MPS-H levels over time (range from 7.7–19.2 ng/mL), was suggestive of ongoing disease-free status.Patient 2 Figure  is a mal4N0M0 SCC of the hypopharynx. She was severely malnourished with multiple medical problems and thought to be a poor surgical candidate. She decided to pursue induction chemotherapy (Carboplatin and Taxol) to be followed by radiation therapy. She underwent three rounds of chemotherapy at 21-day intervals and had a 50% decrease in her tumor size as judged by office endoscopy. She suffered severe GI problems during her therapy, opted not to continue on to radiation, and enrolled in hospice care. The patient did not present for additional follow-up after entering hospice and died three months later. This case illustrates the potential utility of MPS-H as a marker for tumor response to chemotherapy and/or irradiation.Patient 3 Figure  was a feHistological examination of the tumor, surgical margins, and cervical nodes are the current means of determining extent of disease. When surgical extirpation is not undertaken, staging is performed based on a radiological assessment, biopsy, and physical examination. These methods are used either to determine adequacy of resection and the need for adjuvant therapy or to select an alternative primary  therapy, respectively. Limitations of the histologic method include microscopic disease that escapes diagnosis due to a small number of malignant cells, subtle histological changes not classified as cancer, previous treatment effect upon tissues, or pathologic sampling error. CT and physical examination both suffer from modest sensitivity and specificity in detecting many early head and neck neoplasms. As a result, local and regional treatment failures are not uncommon in both surgical and non-surgical treatments of head and neck cancer. This may be due to an underestimation of the tumor burden. This may also be explained by the current diagnostic emphasis upon analysis of structure (microscopic) or anatomic extent of disease, both of which are imperfect, rather than its biologic activity as might be measured by a tumor marker or a functional scanning technique such as FDG-PET.The MPS-H test has been used in conjunction with conventional tumor-specific markers to improve sensitivity and specificity of tumor serodiagnosis . Of all Control groups of healthy volunteers and those with systemic, non-malignant diseases have been studied using the MPS-H test. Statistical analysis has revealed that those without malignancy have MPS-H serum levels less than 10 ng/mL, those with malignancy have levels greater than 20 ng/mL, and those with bony metastasis have levels in excess of 100 ng/mL . AdditioSerum samples from prostate carcinoma patients with high levels of MPS-H (>500 ng/mL) have demonstrated the authentic 9.4 kDa MPS-1 protein, at least one protein with sequence homology to the N-terminus of MPS-1, and a high molecular weight precipitation interfering protein.More accurate detection of recurrent SCC of the head and neck by the MPS-H test may provide a new way to improve survival. Physical exam, conventional imaging, and biopsy are the current gold standard to determine recurrence. Currently, FDG-PET is the most promising way to assess early tumor recurrence of the head and neck but is quite expensive ,26. Its In the present study we compared FGD-PET to MPS-H levels in head and neck cancer patients. FDG-PET interpretation and MPS-H level determination were performed independently and blinded from the results of the other test. FDG-PET positive scans were not all confirmed by biopsy in our study. A statistically significant correlation was noted between FDG-PET positive cases and high MPS-H serum levels in head and neck cancer patients. MPS-H and FDG-PET agreed in 103 out of 183 cases. In 12 cases MPS-H was elevated but no cancer was found by FDG-PET, suggesting that the patients may have had an early recurrence detectable by MPS-H but not yet by FDG-PET. The 68 FDG-PET positive cases that show low MPS-H levels suggest that some tumors were unable to produce high levels of MPS-H, perhaps due to previous chemotherapy, or that some results represent false positive PET scans. Further study with larger patient groups is ongoing to assess the optimal cutoff levels of MPS-H and the correlation between FDG-PET and MPS-H.Shortcomings of this study include a limited time span, a large proportion of advanced stage cancers, limited controls , and ability to define an absolute cut off value for normal vs. abnormal. We recognize that other factors such as age and other malignancies may effect MPS levels. Future studies will attempt to address and/or control for these issues.Considering the data presented in this paper, which agrees with previous results with other tumor types, and the compelling need to expedite the early diagnosis of primary and recurrent epithelial malignancies of the head and neck, we are further evaluating the MPS-H tests as a tool for diagnosis in a larger group of HNSCC patients. Additionally, we are working to improve the diagnostic technology used to detect MPS-H -29. SincThe authors declare that they have no competing interests.BCS: Principal Investigator, Principal EditorCSH: Data analysis and manuscript preparationCL: Medical student research assistant for projectFD: Member of multidisciplinary head and neck oncology team, recruiter of subjectsVJL: Member of multidisciplinary head and neck oncology team, supplier of PET data, data analysis.PDH: Laboratory technical support with MPS assayThis study was funded by the clinical salaries of BCS, FRD, and VJL. CML and CSH were students at the time of this study. MPS assays were performed at the Department of Veterans Affairs James A. Cochran Medical Center, Molecular Oncology Laboratory, St. Louis, MO. PDH was an employee of the US Government at the time of this research.


Snurf-Snrpn gene. Besides their co-regulated imprinted expression, two observations suggest that the spatio-temporal expression of these genes could also be co-regulated. First, the PWS genes have all been reported to be expressed in the mouse nervous system. Second, Snurf-Snrpn and its associated IC are the most ancient elements of the domain which later acquired additional functional genes by retrotransposition. Although located at least 1.5 megabases from the IC, these retroposons acquired the same imprinted regulation as Snurf-Snrpn. In this study, we ask whether the IC, in addition to its function in imprinting, could also be involved in the spatio-temporal regulation of genes in the PWS domain.The human Prader-Willi syndrome (PWS) domain and its mouse orthologue include a cluster of paternally expressed genes which imprinted expression is co-ordinately regulated by an imprinting center (IC) closely associated to the Snurf-Snrpn and C/D-box small nucleolar RNAs (snoRNAs) MBII-85 and MBII-52 to the expression pattern of the two evolutionary related retroposons Ndn and Magel2, in the developing mouse embryo. We show that these genes have highly similar expression patterns in the central nervous system, suggesting that they share a common central nervous system-specific regulatory element. Among these genes, Ndn and Magel2 display the most similar expression patterns. Using transgenic mice containing the Ndn and Magel2 genes, we show that the transgenic Ndn gene whereas not imprinted is correctly expressed. Search for DNase I hypersensitive sites in the Ndn-Magel2 genomic region and comparative genomic analyses were performed in order to identify potential transcriptional cis-regulatory elements.We compared the expression pattern of Ndn gene in the PWS domain.These results strongly suggest that paternally expressed genes of the PWS domain share a common central nervous system-specific regulatory element. We proposed that this regulatory element could co-localize with the IC. However, we demonstrate that the IC, if required for imprinted regulation, is not involved in the spatio-temporal regulation of distantly located retrotransposed genes such as the  Genomic imprinting in mammals is a process that leads to the preferential mono-allelic expression of specific genes in diploid cells, depending on whether they are inherited from the sperm or from the oocyte. To date, approximately 70 mammalian imprinted genes have been identified which map to at least 11 regions of the mouse genome . ImprintSNURF-SNRPN, NDN, MAGEL2, MKRN3 and the C/D-box small nucleolar RNAs(snoRNAs). In humans, mini-deletions, upstream the SNURF-SNRPN transcriptional unit, have led to the characterization of an IC which coordinates their imprinted expression [SNURF-SNRPN promoter, has been shown to be required for maintaining the paternal imprint [Snrpn promoter is not required for genomic imprinting [Snurf-Snrpn exon 1 to Snurf-Snrpn exon 7 unit also leads to silencing of the paternally expressed genes, indicating that both the position of the IC and its role in the coordinate imprinted expression of genes is conserved between human and mouse [The Prader-Willi syndrome (PWS) domain on human chromosome 15q11-q13 and its ortholog on mouse chromosome 7C-D1 are large chromosomal domains containing paternally expressed genes [pression . One sub imprint . Its del imprint ,6. In mind mouse ,9.Snurf-Snrpn locus and its associated IC would be the ancestral imprinted transcriptional unit of the domain. The other genes, which are intronless, would have later and sequentially been acquired by retrotransposition and adopted the same imprinted regulation. Altogether, these data suggest that a spatio-temporal co-regulation of PWS genes could exist and have been acquired through the evolution of the domain. Thus, it is tempting to speculate that the cis-regulatory element(s) initially involved in the spatio-temporal regulation of the Snurf-Snrpn locus might influence the spatio-temporal regulation of retrotransposed genes in the PWS region. The IC could be a good candidate to play this role.Recently, examination of tissue-specific pattern of mRNA expression at a genomic scale allowed the identification of several chromosomal regions harbouring tissue-specific co-regulated genes defined as regions of correlated transcription (RCTs) . NoticeaSnurf-Snrpn, MBII-85, MBII-52, Ndn and Magel2 in the developing mouse embryo. We show that their expression in the embryo is restricted to neural tissues and that these genes display strikingly similar expression patterns in the central nervous system. Second, we created transgenic mice with a BAC containing the Ndn and Magel2 genes. We showed that the Ndn transgene is not imprinted but is correctly expressed in the developing embryo. These results demonstrate that if the IC is required for imprinted regulation, it is not involved in the Ndn spatio-temporal regulation. Finally, since we have shown an almost identical spatio-temporal expression profile of Ndn and Magel2 in the developing embryo, it suggests that these two genes might share common cis-acting regulatory elements which should be present in the BAC transgene. We therefore searched for DNaseI hypersensitive sites in the Ndn-Magel2 genomic region and performed comparative genomic analyses in order to identify potential transcriptional cis-regulatory elements.In this study, we investigate this hypothesis. First, we compared the expression pattern of the Snurf-Snrpn, MBII-85, MBII-52, Ndn and Magel2 by in situ hybridization in the developing mouse embryo . As previously reported for Ndn [Ndn and Snurf/Snrpn are both expressed at high levels in cranial and dorsal root ganglia, sympathetic and parasympathetic ganglia, structures in which MBII-85, MBII-52 and Magel2 trancripts are expressed at much lower levels or not at all  and in some non-neuronal neural crest cells derived structures such as the branchial arches  or even exclusively  in the mouse developing nervous system at E12.5 and E13.5, with strikingly similar identical patterns in the central nervous system . More divergent expression patterns were observed in the peripheral nervous system , some genes being expressed at high levels (Ndn and Snurf-Snrpn) and the others being expressed at low levels or not at all . Our in situ hybridization data suggests the presence in the PWS domain of a central nervous system-specific neural element which coordinates the PWS genes expression.In conclusion, all the PWS genes that we studied were expressed mainly  containing both genes to generate transgenic mice . Whether maternally or paternally inherited, the transgenic Ndn gene was expressed with the same tissue specificity as the endogenous Ndn gene  and wild type mouse embryos, to compare the transgenic and endogenous profile of Ndn expression. Transgenic Ndn transcripts were detected in embryos from E10.5 as endogenous Ndn transcripts, and in the same structures, namely the central and peripheral nervous system  which regulatory potential can further be assessed experimentally [BglII- or BamHI-cut DNA with a series of probes described in Fig. Ndn gene  and of one site upstream the Magel2 gene (HS5)  and strong DNase I hypersensitive sites (HS1 and HS2) in the Ndn promoter region of the paternal allele . The presence of DNase I hypersensitive sites HS1/2, HS3 and HS4 is therefore closely linked to the transcriptional activity of the Ndn gene since they were present on the paternal allele and only in Ndn-expressing tissues.Hybridization ele Fig. . HybridiBamH1-cut DNA with probe 6 or BglII-cut DNA with probes 7 and 8 revealed one relatively strong DNase I hypersensitive site (HS5) 3.5 kb upstream the Magel2 gene, which was present on both parental alleles and co-localized with a polymorphic BglII site present on the CAST/Ei allele. HS5 was localized 1 kb upstream newly isolated full length Magel2 ESTs  [Magel2 and/or Ndn are not expressed such as the adult kidney (data not shown) which suggests that this DNase I hypersensitive site is not linked to the transcriptional activity of these two genes.Hybridization of brain M013779) . HS5 wasNdn-Magel2 intergenic region (excepted HS5). Multiple faint hybridization signals could however be detected with probes 3 and 4 on BglII-cut DNA (data not shown) and with probe 5 on BamH1-cut DNA, revealing regions of low DNase I hypersensitivity in the Ndn-Magel2 intergenic region . We were not able to precisely localise the HS6 site(s) as well as its (their) parental origin.No prominent DNase I hypersensitive site were detected in the ion Fig. . FinallyNdn gene to the Magel2 extremity of BAC109. Several CNS defined as orthologous sequences greater than 100 bp and greater than 70% identity [Ndn gene (region A), in the intergenic Ndn-Magel2 region (region B) and in a 2.8 kb upstream the Magel2 gene (region C). None of the hypersensitive sites identified in this study mapped to region A, B or C. HS1/2 which are closely linked to the transcriptional activity of the Ndn gene are localized in a CNS immediately upstream the Ndn gene. HS5 co-localized to an isolated CNS localized 0.7 kb upstream the highly conserved C region and around 1 kb upstream the newly described full length Magel2 ESTs. However, the functional significance of HS5 in the Magel2 and/or Ndn gene transcriptional regulation remains to be determined since this site was neither allele nor tissue-specific. It should be noted that there was almost no conservation of sequence in the region downstream the Magel2 gene. In contrast, the intergenic Ndn-Magel2 region was relatively well conserved and regions of low DNase I hypersensitivity were detected in this region which might have functional importance.We next mapped HS1 to 6 DNase I hypersensitive sites on a mouse/human sequence alignment Fig. . The avaidentity  were fouSnurf-Snrpn, MBII-85/-52 snoRNAs, Ndn and Magel2 expression profiles brought new information on the transcriptional regulation of these genes. Snurf-Snrpn, the snoRNAs MBII-52 and MBII-85 and the distantly located Ndn and Magel2 genes were expressed in the mouse developing nervous system at E10.5 and E13.5 with strikingly similar patterns in the central nervous system.Study and comparison of More divergent expression patterns were observed in the peripheral nervous system and non-neuronal tissues.Snurf-Snrpn, MBII-85 and MBII-52 snoRNAs were exclusively expressed in the developing nervous system, and in identical structures in the central nervous system. Marked differences of expression between Snurf-Snrpn and the snoRNAs were found in cranial and dorsal root ganglia, structures in which Snurf-Snrpn was highly expressed whereas the snoRNAs were expressed at very low levels (MBII-85) or not at all (MBII-52). These results reinforce the proposal that the snoRNAS derive from the processing of long primary transcripts initiated at the U exons, upstream Snurf-Snrpn [Snurf-Snrpn was previously reported to be ubiquitously expressed in adult tissues [Accordingly, rf-Snrpn  . Particularly striking was their identical pattern of expression in the E10.5 embryo. At later developmental stages, Magel2 expression domain became more restricted but remained included in Ndn expression domain. Magel2 transcripts were however detected at very low levels in almost all the structures in which Ndn was expressed, including non neuronal structures such as the mandibular component of the first branchial arch, the tongue and the muscles. Magel2 transcripts have been reported to be particularly instable due to the presence of AU-rich elements (ARE) in their 3' untranslated region [As previously reported ,11, Ndn d region  and thisNdn and Magel2, specifically, could share additional regulatory elements.Our expression analysis in the mouse embryo indeed suggests the existence of a central nervous system-specific neural regulatory element coordinating the PWS genes expression. Snurf-Snrpn locus with its associated IC has been proposed to be the most ancestral transcriptional unit of the domain [Ndn, Magel2, Mkrn3 and Frat3) would have later been acquired by retroposition and/or local cis-duplication of retroposed genes and would have adopted the same transcriptional regulation as the Snurf-Snrpn transcriptional unit. Unless retroposons arise from reverse-transcriptase-mediated processing of aberrant or alternative transcripts including endogenous promoter elements, their functionality is dependent upon regulatory elements present at their site of insertion. Retroposons in the PWS region could have therefore adopted the Snurf-Snrpn regulation both for imprinted and spatio-temporal expression. Finally, the fact that paternal deletions of the IC abolish the expression of all the PWS genes [We further emitted the hypothesis that the central nervous system-specific regulatory element could be physically associated to the PWS IC because of the proposed evolutionary history of the PWS domain and because the IC is involved in coordinating long range chromatin modifications leading to imprinted expression of genes in the whole domain. The e domain ,13. OtheWS genes ,7 also sNdn does not rely on the IC for its spatio-temporal expression. Ndn was correctly regulated from the BAC transgene lacking the IC both in the developing embryo and in adult tissues. These results demonstrate that the cis-regulatory sequences involved in both the developmental and tissue-specific expression of Ndn were present in the transgene and were not associated to the IC. The fact that the IC was not involved in the spatio-temporal regulation of the Ndn gene does not however exclude its putative involvement in the spatio-temporal regulation of the Snurf-Snrpn transcriptional unit. No transcriptional regulatory elements or sequences excepted those involved in the imprinted expression [Snurf-Snrpn transcriptional unit.Our study did not however confirm that the IC might coordinate the spatio-temporal expression of PWS genes. Our transgenic analysis clearly showed that pression  have yetcis-regulatory sequences involved in both the developmental and tissue-specific expression of Ndn were present in the transgene and since we showed that Ndn and Magel2 expression was coordinated both in the central nervous system and in some non neuronal tissues of the mouse embryo, we searched for Ndn and Magel2 regulatory elements in the BAC109 genomic sequences. Ndn and Magel2 are two evolutionary related retrotransposons, which belong to the MAGE D gene family [cis-duplication event . Their close genomic localisation, within 30 kb in the mouse genome, would however be in favour of this second hypothesis. The co-expression of Ndn and Magel2 could therefore result from regulatory elements sharing and/or duplication. Intraspecies genomic comparisons of mouse or human Ndn and Magel2 upstream sequences did not reveal any significant homology which does not exclude the possibility that one of these two genes arose by a cis- duplication. Our experimental search for regulatory elements elements in the Ndn/Magel2 genomic sequences was limited to sequences available in the databases and was therefore not exhaustive. However, we found several DNase I hypersensitive sites  linked to Ndn transcriptional activity and associated to the Ndn promoter. Previous transgenic studies in the zebrafish using a series of hybrid transgenes containing various mouse Ndn promoter sequences associated to the reporter LacZ gene suggested that the mouse Ndn promoter from -845 bp to +63 bp functioned in the zebrafish embryo in a temporal, spatial and tissue-specific manner [cis-acting element driving the neuronal-specific expression was located into an 87 bp sequence from -173 to -87 bp of the Ndn gene which exactly co-localizes with HS1/2. No identified transcriptional factor brain-specific binding site could however be identified in this sequence. Deletion of promoter sequences in Ndn-KO mutant mice [Magel2 transcriptional regulation  which suggests that if the coordinated Magel2 and Ndn expression results from the sharing of a putative enhancer, this enhancer is not associated to Ndn promoter sequences.Since the e family . Phylogec manner . In partant mice  did not NDN gene has been shown to be expressed in a larger panel of tissues than the mouse Ndn gene [Magel2 transcriptional unit, showing the existence of a 2.5 kb highly conserved region upstream the described Magel2 gene. Identification of full length embryonic and brain specific Magel2 ESTs including this region further confirms that this region belong to the Magel2 transcriptional unit.The human Ndn gene ,24 but oNdn gene   antisense RNA  initiated in (or including) the 5' part of Ndn coding sequence and extending in Ndn upstream sequences has recently been described and according to the authors, this antisense transcript could be involved in the imprinted expression of Ndn mRNA [Ndn CDS -and therefore the region in which the antisense transcript is initiated- was deleted [LacZ gene which replaced the Ndn was monoallelically expressed, further strengthening an absence of role of this hypothetical antisense transcript in Ndn imprinting.As expected, our transgene which was physically separated from the IC was not imprinted. When outside of its natural genomic context, Ndn mRNA . This tr deleted , the LacNdn gene, although not imprinted anymore, can still be correctly expressed in the developing nervous system of the mouse embryo. The BAC transgene therefore contained the regulatory elements needed for the spatio-temporal expression of Ndn (and most likely Magel2) in the developing nervous system. Three hypotheses can be made: 1) the PWS genes co-expression that we observed is incidental, 2) the element(s) localised in genomic sequences surrounding the Ndn/Magel2 and which regulate(s) Ndn (and Magel2) expression could also regulate other PWS genes, 3) several central nervous system-specific regulatory elements resulting from duplication events might be present in the PWS domain. The evolutionary history of the PWS domain is more in favour of the third hypothesis but will need further investigations to be confirmed.Our analysis strongly suggests the existence of a central nervous system-specific regulatory element which would coordinate expression of the PWS genes in the developing mouse embryo and we proposed that this element could be associated to the IC. However, our transgenic studies clearly demonstrate that when physically separated from the IC, a transgenic M. musculus castaneus (CAST/Ei) male mice from the Jackson Laboratory. Mice carrying an Ndn null mutation on a 129/Sv genetic background [Adult C57BL/6 and C57BL/6 X CBA mice were purchased from IFFA CREDO, and ckground  and tranin situ hybridization experiments for the study of PWS genes expression were performed on C57BL/6 embryos. In situ hybridization was performed on 14 μM paraformaldehyde-fixed cryosections with antisens digoxigenin-labeled riboprobes. Sections were washed in 1X PBS, treated with RIPA buffer , post-fixed in paraformaldehyde, acetylated and further washed in PBST before the hybridization step. All hybridization and post-hybridization washes were performed at 70°C. Hybridization was performed in 50% formamide, 5X SSC, 5X Denhart's solution (Sigma), 0.5 mg/ml herring sperm DNA, 0.25 mg/ml yeast RNA. Washes were performed in 50% formamide, 2X SSC, 0.1% Tween 20. Digoxigenin labelling was detected using anti-digoxigenin Fabs  coupled to alkaline phosphatase and NBT/BCIP (Sigma). The Ndn riboprobe (290 bp) hybridizes to the 3' UTR of the Ndn mRNA . The Magel2 riboprobe (318 bp) hybridizes to the 3' part of the Magel2 ORF . The snoRNAs MBII-85 and MBII-52 riboprobes were synthesized from plasmids given by J. Cavaillé. The Snurf riboprobe (277 bp) hybridizes to U/Snurf-Snrpn transcripts and recognizes sequences from the 3' end of the Snurf exon 1 to the 3' end of the Snurf exon 3 .All Ndn probe, and contains a 103 kb NotI insert comprising the Ndn and Magel2 genes. The Ndn gene is localized in the center of BAC109, the EagI intragenic site being situated 54 and 50 kb away from the vector NotI sites and the 3' extremity of the Magel2 gene is localized 17 kb from one of the vector NotI site  was performed on embryos or adult mice inheriting the transgene either paternally or maternally, on a Ndn null background. These embryos or adult mice were obtained by mating transgenic males carrying the Ndn null mutation to wild type C57BL/6 females (Tg92pat-Ndn+/-pat) or transgenic females to males carrying the Ndn null mutation (TgBAC92mat-Ndn+/-pat).BAC109 was isolated by hybridization of mouse bacterial artificial (BAC) high-density membranes  with a genomic M. musculus C57BL/6 females and M. musculus castaneus males (B6 x CAST/Ei) F1 or from inbred C56BL/6 mice (B6). Nuclei were isolated from adult mouse tissues as described [2, 0.1 mM EGTA, 15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.2 mM PMSF, 0.4 mM CaCl2, 5% glycerol) at a concentration of 5.107 nuclei/ml. Increasing amounts of DNase I  from 0 to 100 units diluted in 5 μl of DNase I digestion buffer were added to 95 μl aliquots of nuclei. After a 1 min incubation at 25°C, the digestion was stopped by addition 100 μl of stop solution  and samples were incubated overnight at 37°C. Genomic DNA was purified by multiple phenol/chloroform extractions and precipitated with ethanol. After resuspension, the DNA was digested with the appropriate restriction enzymes, separeted by gel electrophoresis, and transferred to Hybond N+ membranes. The membranes were hybridized by random hexamer- radiolabeled probes, in church solution , at 65°C. Filters were washed three times in 0.2X SSC, 0.1% SDS at 65°C and exposed to X-ray film at -70°C. When necessary, blots were stripped by incubation in 0.1X SSC, O.1% SDS at 95°C. Most probes were prepared by PCR amplification of BAC109 sequences, using the following primers: Probe 1: S-5'-AGATCTGAAGACATAATG-3', AS-5'-GCTCTCCATTTCTAT TAGGTC-3'; Probe 2:S-5'-ATAAGTATTTGGTACTTTCAC-3', AS-5'-TGCTAAGTGCCTACACTGAG3'; Probe 3: S-5'-GAGCGAAACTATTCTGACAG3', AS-5'-AAGCTTCCTCCTCTATGGCAA-3'; Probe 4: S-5'-GATTTCTGCTAAGATTGG-3', AS-5'-ATGTTCCCTCTAGAAACC-3'; Probe 6: S-5'-AGTTAGAGACAAGCCTAG-3', AS-5'-TTCTGGGATGTCTCAGGA-3'; Probe 7: S-5'-GCATTTTGAGGAAGTACCCA-3', AS-5'-CATGGCCATTTCTAACTGTG-3'; Probe 8: S-5'-CCAAGGAGCTTGGAGGGC-3', AS-5'-CTCGTAGAGTGCGGCCAA-3'; probe 9: S-5'-ACATCAATAGTTTGATAC-3', AS-5'-GGGTGTGGCTGTGCATTGTT-3'Tissues were dissected either from inter-specific F1 hybrids between escribed . Nuclei FW drafted the manuscript, designed the study, and carried out the PWS genes expression analysis, the transgenic analysis and the search for transcriptional regulatory elements. ELM contributed to the PWS gene expression analysis. NR carried out molecular studies on the transgenic lines and participated to the search for DNase I hypersensitive sites. LD was involved in the design of the transgenic studies. MR injected the transgene and established the transgenic lines. FM coordinated the project and participated to the manuscript draft.


Acute HIV-1 infection induces massive activation of CD8+ T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8+ T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8+ and CD4+ T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8 + T-cells, resulting in replicative senescence. This may be part of the mechanism through which HIV-1 exhausts the capacity of the immune systemHIV-1 infection induces activation and differentiation of CD8 In commviraemia . Continuviraemia  and destg cells)  may explnfection , HIV-infnfection  and redunfection , which r+ T-cell differentiation, and outcome in the context of HIV-1 pathogenesis. We report here that T-cell activation and differentiation are closely related, and that HIV-1 induces immune activation directly and indirectly, which results in differentiation of CD8+ T-cells towards replicative senescence.Increasing evidence suggests an association between high levels of immune activation and poor outcome in HIV-infected individuals , althoug+ count above 500 per milliliter and mean viral load of 104 copies/ml), and chronic infection with signs of disease progression . In addition to analysing whole CD8+ T-cell populations in these individuals, we have used a panel of tetramers to study the phenotypic evolution of CD8+ T-cells specific for HIV, cytomegalovirus (CMV), Epstein–Barr virus (EBV), and influenza. Although this approach focuses on a limited number of viral epitopes (restricted by the number of tetramers available), it remains the only way to avoid stimulation of the cells in order to detect them , which may alter cellular phenotype and does not enable the detection of all cells.Our study involved the analysis of two distinct groups of HIV-1-infected individuals. On one hand, we performed a longitudinal analysis of T-cell subsets during acute HIV-1 infection and its resolution. To examine the effect on T-cells of elevated immune activation associated with an episode of vigourous HIV replication , T-cells were studied in individuals during HIV acute infection and later on—postacute—when viral replication was suppressed following the start of antiretroviral therapy (ART) . These d+ T-cells were strongly activated, and, intriguingly, activation of the CD8+ T-cell compartment as a whole was particularly high, reaching to levels of 80%–90%, in contrast to CD4+ T-cells, which show much less activation  expression during acute infection (p = 0.03), in keeping with activation-related proliferation of this subset, as previously described (+ between acute and postacute infection stages was observed within the naïve CD8+ T-cell subset (CD62L+/CD45RA+) and antigen-experienced CD45RA+ , intermediate (CD28−/CD27+), and late (CD28−/CD27−) differentiated subsets  CD8+ T- T-cells B. This i subsets . While bnfection C, sugges+ T-cells specific for non-HIV viral antigens in donors with suitable human leukocyte antigen (HLA) type , both CMV- and EBV-specific CD8+ T-cells displayed significant levels of activation exclusively during acute HIV infection, compared to chronic infection (p < 0.002) , strongly suggests that the stimulation of these cells associated with HIV-1 infection is due to reactivation of pathogens such as CMV and EBV, rather than as a result of bystander activation. Overall, these data show that HIV-1 infection leads to activation of antigen-experienced CD8+ T-cells at early stages of differentiation, both in direct (HIV-specific) and indirect (HIV-nonspecific) manners.Surprisingly, from the analysis of CD8< 0.002) . Activatker Ki67 E. Plasma T-cells D, in con+ T-cells by dendritic cells (DCs), which represents a useful model to analyse the generation of antigen-experienced CD8+ T-cells. This system is based on the existence in normal human donors of a significant number of naive CD8+ T-cells ; moreover, the half-life of class I MHC–peptide complexes on mature DCs is rather short (+ T-cell population (+ T cells (+ T-cell differentiation observed ex vivo (+/CD27+/CD62L+/CD45RA+/granzyme A−) at day 0 (data not shown), antigen-primed cells lost sequentially expression of CD28 and CD27 , which er short ; therefopulation A. This rpulation B. The pr T cells C. Increa T cells C. The an ex vivo : startinand CD27 D. Followhe cells E. These + T-cells. Despite that the majority of HIV-specific CD8+ T-cells are usually found at an intermediate stage of differentiation  and the size (percentage of tetramer-positive cells in the whole CD8 population) of a variety of HIV-specific CD8+ T-cell populations in several donors revealed a correlation between these two parameters   EBV-specific CD8+ T-cells became further differentiated  B. The dientiated B. This infection . Taken t+ T-cell population along the pathway of differentiation during HIV-1 infection was analysed in a cross-sectional study of individuals at different stages of infection. It revealed an increase in the proportion of highly differentiated CD8+ T-cells associated with HIV disease progression   equivalent to those observed in antigen-experienced CD8+ T-cells from the elderly  C. In addsespairs , until tsespairs . Shortensespairs D so that elderly . All tog ex vivo . However ex vivo .− CD8+ T-cells do proliferate . Overall, the accumulation of highly differentiated CD8+ T-cells in HIV infection goes along with reports of reduced proliferative capacities and shorter telomere length characterising the T-cells of the HIV-infected individual . Converging evidence suggests that a reduction of replicative potential occurs with extensive T-cell division and differentiation. Differentiation towards late stages (CD28−/CD27−/CD57+) is strongly associated with the display of characteristics of replicative senescence, which may have an impact on viral control. The relevance of perforinhigh late-differentiated T-cells in conferring protective immunity is controversial. For instance, + T-cells and years of AIDS-free survival. However, it remains to be determined whether late-differentiated CD8+ T-cells would simply accumulate in these individuals with chronic infection over time, whilst playing no role in delaying disease progression. Overall, there is confusion regarding the ideal functional and phenotypic profile of a “protective effector cell.” Protective immunity has recently been associated with the proliferative capacity of virus-specific CD8 T-cells in the mouse model (+ T-cells that maintain a strong proliferative capacity following in vitro stimulation (cells defined mainly as CD45RO+/CD28+/CD27+ early-differentiated cells). In this study, the proliferative potential of these cells was coupled to strong perforin expression, suggesting that early-differentiated cells  are ablgression . One maygression . More inThe elevated and chronic stimulation induced by HIV-1 may result in the exhaustion of the capacity to generate new T-cells , while t+ cells , and either a negative HIV-1 ELISA or a negative/indeterminate HIV-1 Western blot. A second sample was analysed at a later timepoint after the start of successful ART  or with evidence of progressive HIV disease . Blood samples were also obtained from healthy adult volunteers. Peripheral blood mononuclear cells (PBMCs) were separated from heparinised blood and cryopreserved for subsequent studies. HLA typing was carried out by amplification refractory mutation system–polymerase chain reaction (ARMS–PCR) using sequence-specific primers as previously described  and San Diego (United States) who were known to have either acute or chronic HIV-1 infection. The relevant local Institutional Review Boards and Ethics Committees approved the study. Subject ages ranged from 23 to 65 y old. Eleven patients with HIV-1 acute infection were selected from a well-characterised cohort in San Diego on the basis of their having an HLA type  for which we could detect virus-specific CD8 ART see . The stuescribed . HLA-typHLA–peptide tetrameric complexes (“tetramers”) were produced as previously described  and inclTM lysis solution (Becton-Dickinson). Cells were washed, fixed, and permeabilised in FACSTM permeabilisation buffer (Becton-Dickinson). After washing, intracellular perforin staining was performed using titrated antibodies. Cells were then washed and stored in Cell FixTM buffer (Becton-Dickinson) at 4°C until analysis. Samples were analysed on a Becton-Dickinson FACSCalibur after compensation was checked using freshly stained PBMCs. Carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling was performed by incubating PBMCs with 5 μM CFSE  in RPMI 1640 for 10 min at 37°C, before quenching with ice-cold RPMI 1640–10% foetal calf serum (FCS) and washing. The cells were then incubated with immobilised OKT3 (10 μg/ml) for 6 d (with or without 20U/ml of IL-2) before staining.FACS stainings were performed as previously described . In brie+ T-cell subsets were sorted ex vivo from freshly isolated PBMCs, on the basis of CD27, CD57, CCR7, and CD45RA expression using a five-color FACS vantage SE (with 98%– 99% purity). For each subset, 0.5 × 105 to 2 × 105 cells were used to measure the average length of telomere repeats at chromosome ends in individual cells by quantitative flow fluorescence in situ hybridisation (FISH), as previously described  and 500 U/ml IL-4  for 5 d. Cells (3 × 105/ml) were stimulated by addition of 1 μg/ml LPS . Antigen-presenting cells were pulsed for 3 h with various concentrations of melan-A–peptide in serum-free medium before incubation with autologous PBMCs at a 1:5 ratio in RPMI 1640–10% FCS. Human rIL-2  was added from day 4 at 10 U/ml, then at 500 U/ml IL-2 when cells expanded. Melan-A-specific CD8+ T-cells were then analysed by flow cytometry over time for up to 50 d.DCs were generated as previously described . MonocytP values above 0.05 were considered not significant.Group medians and distributions were compared by the nonparametric Mann–Whitney test. Associations between variables were determined by the nonparametric Spearman rank correlation test. Associations between variables in different patient groups were determined by simple linear regression or ANOVA test.


I (stress intensity factor) diagram. Slow crack growth in ceramics is attributed to corrosion assisted stress at the crack tip or at any pre-existing defect in the ceramic. The combined effect of high stresses at the crack tip and the presence of water or body fluid molecules (reducing surface energy at the crack tip) induces crack propagation, which eventually may result in fatigue. The presence of a threshold in the stress intensity factor, below which no crack propagation occurs, has been the subject of important research in the last years. The higher this threshold, the higher the reliability of the ceramic, and consequently the longer its lifetime.Slow crack growth can be described in a v (crack velocity) versus KWe utilize the Irwin K-field displacement relation to deduce crack tip stress intensity factors from the near crack tip profile. Cracks are initiated by indentation impressions. The threshold stress intensity factor is determined as the time limit of the tip stress intensity when the residual stresses have (nearly) disappeared.We determined the threshold stress intensity factors for most of the all ceramic materials presently important for dental restorations in Europe. Of special significance is the finding that alumina ceramic has a threshold limit nearly identical with that of zirconia.fast crack growth. Considering two ceramics with identical threshold limits, although with different critical stress intensity limits, means that both ceramics have identical starting points for slow crack growth. Fast catastrophic crack growth leading to spontaneous fatigue, however, is different. This growth starts later in those ceramic materials that have larger critical stress intensity factors.The intention of the present paper is to stress the point that the threshold stress intensity factor represents a more intrinsic property for a given ceramic material than the widely used toughness (bend strength or fracture toughness), which refers only to  Slow crack growth in ceramics is attributed to corrosion assisted stress at crack tips or at any defect pre-existing in the ceramic [Slow crack growth is most suitably described in a  ceramic . The comI > KIc) initiates fast catastrophic crack growth, followed by the deterioration of a dental or a body restoration machined from ceramics. The presence of stress intensities above a threshold value (KI > KI0) initiates SCCG in ceramics, followed by a slow, however continuous, erosion of the strength of a restoration which also may result in final fatigue. In an early stage of ceramic research it was believed that this lower limit for SCCG is very close to zero. In the mean time, however, one has learned that for most ceramic materials the lower limit for SCCG is significantly larger than zero. Indeed, it may even be just below KIc.The presence of stress intensities above a critical value . We use soda lime glass as a well characterized standard and silicon nitride as important in the general field of ceramics.There are several methods available and in the literature extensively described how the threshold limit can be measured. The feasibility of these measurement procedures is mostly demonstrated with the help of soda lime glass as a brittle solid model.G = W = 2γ, where G is the energy release rate, W is the Dupré work of adhesion, and γ is the surface energy. If G <W the crack should retract and heal; otherwise it should repropagate [v G characteristics  at specified chemical concentrations and temperatures, which can describe observed v-G dependencies.In principle, the proper test for existence of a threshold lies in the observation of reversibility of crack growth. The threshold can be regarded as a Griffith quiescent point, where forward and backward fluctuations just balance, i.e., the mean velocity of the crack tip becomes zero. The forward and backward fluctuations take place over discrete energy barriers definable as ropagate ) calcula-14 m/s. Then one can extrapolate from the vertical branch of the function to the zero velocity limit on the stress intensity factor axis KI, with the intersection KI equal to KI0 [One common experimental method to determine the threshold limit is to measure slow crack growth rate down to velocities as low as 10l to KI0 .Another method to determine the aforesaid threshold limit is the "interrupted static fatigue test" (ISF-test) . For a bv - K diagram, the threshold is determined as described above. For further details refer to [Another method uses a side grooved specimen with a crack propagating along its length, and under a bending condition similar to four point bending. The crack velocity can be obtained from the rate of load relaxation at constant displacement and the initial crack length. Having established the refer to .Other methods may be characterized by the phrase "decay of residual stress" . Here, ttip via the COD for different times after indentation [The current method of measurement used, however, is based on indentation cracking, analogous to other studies also utilizing flaw initiation for starting the test -13. Afteentation .Using a micro-hardness testing machine, a Vickers indentation is made on the carefully polished surface of a sample of the ceramic to be investigated. Radial cracks emanate from each of the four indentated corner sources.-1 mbar). Hence, there is no longer need to sputter the samples with a gold or carbon layer. Our initial attempts to measure the crack opening displacement (COD) showed that sputtering resulted in blurring the crack banks or even partly hiding the crack. Thus, we abandoned those attempts and started again when the ESEM was available. Before the availability of the ESEM it was nearly impossible to precisely measure crack profiles at submicrometer resolution which, however, is mandatory.To determine the stress intensity present at the crack tip due to the indentation, the near crack tip profile is determined using a scanning microscope . A specific feature of this technique is that it is carried out at a moderate vacuum ;(v = Poisson's constant) being.The measured profiles can be attributed to the crack opening displacement (COD) near the crack tip . The neaKtip is balanced by the toughness of the material KIc :and by re-arrangement:x ≤ 20 μm), a linear relationship is experimentally observed between u(x)2 and x. Ktip can then be calculated from a regression analysis as the slope of a straight line, provided E' is known (see below).If data are taken sufficiently close to the crack tip  and it is plausible to assume Ktip(t→∞) ≈ KI0. Therefore, in the present work, because of slow crack growth, we take the threshold value of the stress intensity factor as the time limit of the slowly decreasing Ktip value. Provided that a suitable high resolution scanning microscope is at hand, there is no need of sputtering the samples, and the presently utilized method is very simple. A potential shortcoming, however, is that this method may need many months or even years until the residual stresses are relaxed and the threshold value is reached.The residual stresses close to the crack tip initiated by the indentation impression gradually decay over time Ktip(t→∞) = KI0 assumes ideal behavior. In real ceramics, especially polycrystalline and composite materials, the crack tip may be shielded from residual load by micro structural elements, which toughen the material in the region just before the crack tip [The authors concede that they have chosen to consider a somewhat ideal situation since the assumption rack tip . This beWe carried out ESEM analyses of crack profiles after 1 hour and then after up to 420 days, at 5 dates distributed over the whole time interval Fig. . After i2O3, coarse grained, load of indention 9,9 kg, Young's modulus 350 GPa , Cerec Mark II, 4 kg, 69 GPa, HiCeram, 6,9 kg, 107 GPa, VMK 95, 4 kg, 91 GPa , Cercon Base, 7,9 kg, 210 GPa, CergoGold, 4 kg, 70 GPa , Dicor, 2 kg, 74 GPa , Empress 1, 5,9 kg, 67 GPa, Empress 2, 5,9 kg, 96 GPa , Lava, 8 kg, 210 GPa , Soda lime glass, 2 kg, 73 GPa , Si3N4, 6 kg, 289 GPa and hipped 5%Y2O3-Zirkon, 8 kg, 210 GPa.We determined the threshold stress intensity of the following ceramics (Soda lime glass and veneering ceramics as reference): Al2 72.65, Al2O3 0.28, MgO 3.98, CaO 8.84, Na2O 13.79, K2O 0.19, other 0.27.The constitution of the soda lime glass was SiOx (2 μm <x < 23 μm), analyzed with the help of Eq. 1'. A linear relationship is observed, from which the value of Ktip (t = 48 h) = 0,90 MPa√m was easily and precisely deduced. Fig. Ktip values determined in an analogous manner for nine examples out of the thirteen investigated ceramics. The gradual decrease of Ktip(t) due to decaying stress intensities at the crack tip becomes apparent. The manner in which Ktip(t) decreases suggests an exponential relationship, as the decrease appears to be linear on a logarithmic scale. The truncation of the measurements after about 104 hours (for reasons of feasibility) appears somewhat arbitrarily, and it cannot be excluded that a further decay, although very small, may have been missed. Note that due to the apparent exponential relationship, the overestimation of the threshold value KI0 due to the truncation after 104 hours becomes smaller and smaller with time. We plan to do further measurements after another interval of 104 hours (417 days). Considering the mathematical aspect, 105 hours (11+ years) would make more sense; but such a long interval is obviously not practicable. As already mentioned, this time constraint is a decided disadvantage of our current method to determine the threshold value.As examples, Fig. KI0= Ktip(t→∞). Fig. KI0 values in comparison with their KIc counterparts.Being aware of the these limitations, and having in mind the neglected possible crack tip shielding as discussed above, we identify KIc is the lower limit for (fast) catastrophic crack growth. Stress intensities exceeding this limit cause fast crack growth at supersonic velocity, and eventually result in destruction of ceramic components. This kind of destruction, however, is not the most common or important, since it can be avoided by strictly limiting the stress intensities existing throughout a component by a suitable shape of construction.KI0 is the upper limit of stress intensities for absence of crack growth and the lower limit for (slow) subcritical crack growth (SCCG). Limiting stress intensities such that they stay always below KI0 means infinite life time for a component, since SCCG becomes irrelevant. Hence, the most favorable characteristic stress intensity values are obvious: KIc as high as possible and KI0 as close as possible to KIc. Such a selection minimizes the extension of the interval in which subcritical crack growth can take place, and it maximizes resistance to catastrophic crack growth due to overloading. Fig. KI0/KIc. Favorable ceramics within their class of toughness are situated at the right hand side of the chart. Note, however, that a perfect ceramic material dependent on the focused area of application has not only a favorable  stress strength relationship but also a high KIc value.KIc zirconia, of course, has properties superior to alumina.At first glance zirconia may seem to be a ceramic material superior to alumina, since it has a critical stress intensity factor Fig. : meaningIt becomes apparent that at moderate stresses alumina and zirconia may be equally suitable choices, and other criteria may become important for favoring the one or the other material. Such reasons may be the ease of shaping, questions of color, ease of veneering, esthetic considerations, availability, and other circumstances.n of SCCG of both ceramics are high (in principle meaning slow SCCG), and the answer to the question of which of the materials has the larger exponent depends on whether static or cyclic behavior is addressed: nstatic = 39 vs 104 and ncyclic = 28 vs 16 for Al2O3 and Y-PSZ, respectively [There is one other aspect to be considered when comparing zirconia and alumina. The exponents Zirconia is known to be sensitive to humidity, which is a particular important issue when prosthetic and orthopedic applications are considered. It is known that yttria stabilized zirconia ceramics can be destabilized during the process of steam sterilization. This is due to hydrothermal transformation, resulting in surface roughening of the zirconia ceramic femoral heads. These femoral heads may also undergo slow degradation during long term implantation in the human body. This low temperature degradation does not become significant before several years, but it does raise the question of the use of zirconia for load bearing systems . In concNote that concerning the sensitivity to humidity, there is a notable difference between ceramics for dental or for orthopedic applications. Ceramics for dental applications are often veneered by a different ceramic, which means that there is a protective shield against humidity attacking from outside of the ceramic tooth .Fig. Ic = 1.17 ± 0.08 MPa·√m or KIc = 2.48 ± 0.22 MPa·√m, respectively; and KI0 = 0.83 ± 0.16 MPa·√m or KI0 = 0.94 ± 0.12 MPa·√m, respectively. Again, the critical stress intensity values are largely different, the threshold values, however, are nearly identical. Compare also "Al2O3" with "Lava" and "Cercon".There are some other examples of ceramics for which a large difference in the critical stress intensities is observed whereas the threshold values are very similar. For these ceramics an analogous argument holds, as given above for alumina vs zirconia. From Fig. RM conceived in the study, designed the study and drafted the manuscript. POW and FJ carried out the experimental work. All authors read and approved the final manuscript. All authors contributed equally to this work.


Salmonella spp. with reduced susceptibility to fluoroquinolones have higher than usual MICs to these agents but are still considered "susceptible" by NCCLS criteria. Delayed treatment response to fluoroquinolones has been noted, especially in cases of enteric fever due to such strains. We reviewed the ciprofloxacin susceptibility and clinical outcome of our recent enteric fever cases.Salmonella enterica Serotype Typhi (S. Typhi) and Serotype Paratyphi (S. Paratyphi) blood culture isolates (1998–2002) were tested against nalidixic acid by disk diffusion (DD) and agar dilution (AD) and to ciprofloxacin by AD using NCCLS methods and interpretive criteria. Reduced fluoroquinolone susceptibility was defined as a ciprofloxacin MIC of 0.125–1.0 mg/L. The clinical records of patients treated with ciprofloxacin for isolates with reduced fluoroquinolone susceptibility were reviewed.S. Typhi and S. Paratyphi isolates had reduced susceptibility to fluoroquinolones (MIC range 0.125–0.5 mg/L). All 7 were nalidixic acid resistant by DD (no zone) and by AD (MIC 128- >512 mg/L). The other 14 isolates were nalidixic acid susceptible and fully susceptible to ciprofloxacin (MIC range 0.015–0.03 mg/L).Seven of 21 (33%) Five of the 7 cases were treated initially with oral ciprofloxacin. One patient remained febrile on IV ciprofloxacin until cefotaxime was added, with fever recurrence when cefotaxime was discontinued. Two continued on oral or IV ciprofloxacin alone but had prolonged fevers of 9–10 days duration, one was switched to IV beta-lactam therapy after remaining febrile for 3 days on oral/IV ciprofloxacin and one was treated successfully with oral ciprofloxacin. Four of the 5 required hospitalization.S. Typhi and S. Paratyphi is clinically significant. Laboratories should test extra-intestinal Salmonella spp. for reduced fluoroquinolone susceptibility.Our cases provide further evidence that reduced fluoroquinolone susceptibility of  Salmonella enterica Serotype Typhi (S. Typhi) and Serotype Paratyphi (S. Paratyphi) with reduced susceptibility to fluoroquinolones are common in India and Southeast Asia [ast Asia . Such isast Asia .gyrA gene which encodes the A subunit of DNA gyrase. These mutations lead to resistance to nalidixic acid, a quinolone, and resistance to this agent can thus be used as an indicator of reduced fluoroquinolone susceptibility [Reduced susceptibility to fluoroquinolones most often arises from point mutations in the quinolone resistance determining region (QRDR) of the tibility .Salmonella. Limited published data suggest that treatment of these infections with a fluoroquinolone may result in a delay in clinical response and possibly treatment failure [Although the mechanisms of resistance have been defined, there is still little information on the clinical significance of reduced susceptibility to fluoroquinolones in  failure .Salmonella isolates for nalidixic acid resistance as a marker for reduced fluoroquinolone susceptibility [Salmonella [As a result of this clinical data, NCCLS currently recommends testing of extra-intestinal tibility . Others lmonella  while solmonella .A poor response to ciprofloxacin therapy in a patient with typhoid fever at our hospital prompted us to review retrospectively the ciprofloxacin MICs and clinical outcomes of other enteric fever cases that were treated with a fluoroquinolone.S. Typhi and S. Paratyphi blood culture isolates recovered from patients at The Ottawa Hospital and The Children's Hospital of Eastern Ontario between 1998–2002. Susceptibility testing was performed using NCCLS methods and interpretive criteria [We tested 21 criteria ,6,7. Discriteria .gyrA gene for all isolates with reduced susceptibility to fluoroquinolones to detect nucleotide mutations. PCR amplification and DNA sequencing was performed using primers [gyrA sequence of the fully susceptible S. Typhimurium NCTC 74  [S. Typhi isolates.We sequenced the QRDR region of the  primers  and cond primers . SequencXbaI. Medical records of patients who were treated with ciprofloxacin for infections due to isolates with reduced fluoroquinolone susceptibility were reviewed to determine clinical outcomes.Pulsed field gel electrophoresis (PFGE) was performed on isolates with reduced susceptibility to fluoroquinolone using S. Typhi and 3 of 9 S. Paratyphi) were nalidixic acid resistant by disk diffusion, all with no zones of inhibition. These isolates were all nalidixic acid resistant with an MIC range of 128->512 mg/L, and demonstrated reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.125–0.5 mg/L). The 14 nalidixic acid susceptible isolates by disk diffusion (zone size range 20–25 mm) were also susceptible by agar dilution (MIC range 2–4 mg/L), and none had reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.015–0.03 mg/L). Multidrug resistance was identified in 5 of 21 isolates (24%) with resistance to ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol.Seven of 21 isolates or 33% . Two of 3 S. Paratyphi had identical PFGE patterns, with the third being possibly related (4 band difference) [S. Paratyphi were susceptible to the other antibiotics tested.PFGE of the 7 resistant isolates showed 3 of 4 ference) . All 3 iS. Typhi isolates from cases 1, 3 and 4 were also resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. The Salmonella isolates from the other 2 cases were susceptible to these antibiotics. Of note, 3 of 4 patients with S. Typhi had fever durations well in excess of the expected 95% upper confidence limit of 3.9 days found in a pooled analysis of multiple studies. These studies involved patients with predominantly nalidixic acid susceptible S. Typhi who were treated with fluoroquinolones [Infections with the 7 isolates with reduced susceptibility to fluoroquinolones were all acquired in the Indian subcontinent. Five of the 7 patients with infections due to these isolates were treated initially with ciprofloxacin. The clinical and microbiological findings for these 5 are summarized in Table 1 see . S. TyphS. Paratyphi with reduced susceptibility to fluoroquinolone was judged to be failing ciprofloxacin, since there was no clinical improvement after 3 days of treatment. Although fever of this duration is common, patients with enteric fever given fluoroquinolones may show some clinical improvement by this time. Three days with no amelioration of symptoms has been used as indicator of a poor response to therapy in some studies [The patient infected with  studies ,12. ThusNone of the 5 had a recurrence of bacteremia after antibiotics were discontinued, and none died.Fluoroquinolones have been considered the treatment of choice for enteric fever. As noted, a mean fever clearance time of only 3.9 days  has been reported, which is shorter than for other agents . CalculaS. Typhi and S. Paratyphi isolates acquired in the Indian subcontinent and Southeast Asia [S. Typhi isolates with reduced susceptibility to fluoroquinolones appears to be the same as that described for S. Typhi strains from India, Pakistan, Bangladesh, and Tajikistan [gyrA point mutations in our isolates have also been previously described in isolates from these countries [However, reduced susceptibility to fluoroquinolones jeopardises the usefulness of these agents. It is now seen frequently in ast Asia , where cjikistan . The gyrountries ,3.Salmonella spp., including case reports, animal experiments, and pharmacodynamic modelling [S. Typhi than for those with nalidixic acid susceptible strains. In addition, one third of nalidixic acid resistant infections required re-treatment vs. 0.8% of infections due to susceptible strains. The authors concluded that short courses (less than 5 days) of oral fluoroquinolone therapy should not be used for treating nalidixic acid resistant isolates. Our small series adds to the evidence that there is a delayed clinical response to fluoroquinolone therapy in this setting, and demonstrates that fever may be prolonged even with long courses of fluoroquinolone therapy, and even when given parenterally in some cases.Several lines of evidence support the clinical significance of elevated fluoroquinolone MICs in odelling . In a reodelling , the medUntil results from randomized controlled trials of enteric fever due to strains with reduced susceptibility to fluoroquinolones are available, the best treatment regimen is uncertain. Current options include higher dose and longer duration of fluoroquinolones, 3rd generation cephalosporins and azithromycin, either alone or in combination . If suscWhen fluoroquinolones are given, it is important to bear in mind that two pharmacokinetic/pharmacodynamic ratios are predictive of successful treatment with these agents . These aSalmonella strains that have reduced susceptibility to fluoroquinolones. The current NCCLS guidelines (2004) recommend that all extra-intestinal isolates of Salmonella be tested for resistance to nalidixic acid in order to detect reduced fluoroquinolone susceptibility [It is important that microbiology laboratories have procedures to detect lines 200 recommenSalmonella spp. with reduced susceptibility to fluoroquinolones [There have also been suggestions to change the NCCLS breakpoints to reflect the risk of treatment failure of inolones . This isinolones .Salmonella spp. with reduced fluoroquinolone susceptibility. Not all laboratories routinely perform MICs and many rely on commercially-available automated susceptibility methods that cannot currently detect reduced fluoroquinolone susceptibility. The current NCCLS recommendations to test for nalidixic resistance are easy to implement for routine testing but may have reduced specificity [It is not completely clear at present which of these is the optimal approach for laboratory detection of cificity . LaboratSalmonella spp. with reduced susceptibility to fluoroquinolones is clinically significant. It is important that laboratories test S. Typhi and S. Paratyphi, as well as other extra-intestinal Salmonella isolates, for reduced susceptibility to fluoroquinolones.Given the accumulating evidence, including our own clinical experience, enteric fever due to None declared.RS: participated in the design of study and authored first draft; MD: conceived study, coordinated susceptibility testing, and revised manuscript; AM: conceived study and reviewed clinical data; KR: participated in study design and coordinated pulsed field gel analysis; PJ: reviewed clinical data and helped coordinate susceptibilty testing; CB: carried out molecular genetic studies and participated in sequence alignment; BT: conceived study and supervised study, revised manuscriptAll authors read and approved of the final manuscript.The pre-publication history for this paper can be accessed here:S. Typhi or S. Paratyphi isolates with reduced fluoroquinolone susceptibility provides information regarding clinical course for patients with isolates with reduced fluoroquinolone susceptibility as well as antibiotic susceptibility and gene mutation results for these isolatesClinical and microbiological information for patients treated with ciprofloxacin for Click here for file


Based on sheer numbers, microbes should rule the world. Most don't cause disease, but those that do have the advantage of multiplying and mutating at a much faster rate than any multicellular organism can. So how does a slowly reproducing, trillion-celled organism like a human protect itself? By having the right weapon for the job—and that requires an incredibly diverse arsenal. A new study by a team of researchers from Yale University School of Medicine, Duke University Medical Center, and Mount Sinai School of Medicine demonstrates how the creation of that arsenal depends on a complex series of interactions between key genetic elements and proteins during the formation of the white blood cells called lymphocytes.Two heavy hitters of the immune system—B and T cells—each produce unique protein receptors that specifically recognize and mediate the killing of the variety of potential foreign invaders, or antigens, such as bacteria, viruses, and parasites.  But these lymphocytes are unlike other cells: instead of making proteins from genes they inherited, they custom-make their genes by recombining fragments of their genes into new configurations. This genetic reshuffling process, called V(D)J recombination, yields the diversity of molecules necessary to combat the billions of different antigens they might encounter. The V, D, and J refer to different clusters of DNA sequences that follow specific rules of recombination.While the products of recombination vary, the method does not. The fragments are spliced and then reassembled in a highly regulated process directed and controlled by a stretch of DNA  next to the gene fragment. The recombination process, the researchers show, relies on complex interactions among different parts of the signal sequences and the proteins that regulate them at key steps along the recombination pathway.Each RSS is made up of three components: the nonamer, which controls the ability of proteins to bind to the gene fragments and initiate recombination; the heptamer, which directs the splicing of the gene fragment; and the spacer, which regulates how the gene fragments are recombined. Mutations in the DNA sequence of each of the three RSS components show that all play a critical role in the ability of the gene fragments to recombine appropriately.While it has been established that spacers, as their name suggests, ensure that the space between the nonamer and heptamer is correct, the researchers show that spacers also regulate recombination activity by providing protein-binding sites along the DNA sequences that affect recombination. While the nonamer is the most important determinant of recombination, changes in the spacer, these researchers demonstrate, produced dramatic changes in the ability of the gene fragments to recombine.Past studies have shown that recombination depends on the presence and sequence of specific nucleotides, but the quality of that recombination, the researchers say, can't be understood simply by analyzing those nucleotides in isolation. Generally speaking, highly conserved sequences have functional importance. But it would be a mistake, they suggest, to think that just because a nucleotide sequence isn't highly conserved, it's not biologically important. Using a computer model to predict how different protein-gene interactions affect recombination, the researchers demonstrate that a fuller understanding of the process depends on observing how all these elements—including those that aren't highly conserved—interact throughout the recombination process.


Almost one-third of all penile metastases are detected at the same time as a primary tumor, whereas the remaining two-thirds are detected a mean of 18 months after the discovery of the primary tumor. Cutaneous metastasis of transitional cell carcinoma (TCC) is extremely rare and generally accepted as the late manifestation of a systemic spread.We report the first case of simultaneous penile and lung metastases from a primary TCC of the renal pelvis in a 76-year-old man, that occurred 8 years after a left nephroureterectomy.This case report underscores the importance of physical examinations of the skin of patients who undergo surgical procedures for TCC from bladder as well as from the upper urinary tract, including those seemingly without metastatic disease, because of the possibility of skin and penile metastatic spread. Approximately 300 cases of penile metastases are reported worldwide . SeventyA 76-year-old man with a painful, ulcerative swelling of the glans that had appeared less than 2 months previously was brought to our attention. Physical examination revealed a 3.5-cm fungating lesion involving the upper half of the glans Fig. . The lesThe patient began combination chemotherapy  and external beam radiotherapy to the mass, which promptly relieved the penile pain. At an 8-month follow-up the patient was still alive with no remarkable changes to the pulmonary and lymph node metastases, or the penile swelling.Cutaneous metastases from primary TCC of the urinary system are extremely rare and are generally accepted as late manifestations of systemic spread . SpectorAlmost one-third of all penile metastases are generally detected at the same time as a primary tumor, whereas the remaining two-thirds are detected a mean of 18 months after the discovery of the primary tumor [The optimal treatment of penile metastasis requires a multidisciplinary approach that is correlated with the disease extent. The average survival in patients with penile metastasis is 3.9 months from diagnosis and, with extensive surgery and chemotherapy, a survival of 9.2 months has been reported .This patient was not eligible for platinum-based regimens due to impaired renal function (prior nephrectomy) and advanced age. As a result, he was treated with gemcitabine as a single agent. This treatment has demonstrated objective response rates of 25–29% with minimal toxicity as compared to standard regimens .In conclusion, this case report suggests that particular attention should be paid to physical examination of the skin of patients who undergo surgical procedures for TCC from bladder as well as from the upper urinary tract, including those seemingly without metastatic disease, because of the possibility of skin and penile metastatic spread.The author(s) declare that they have no competing interests.GP drafted the manuscript and coordinated the co-authors. IP, MS, PC participated in the sequences alignment. GM carried out the histological features of the lesion. FF participated in the sequences alignment and coordinated the co-authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: