Abstract Rapid proliferation of CD8 T cells is crucial for adaptive immunity against viral infection. CD8 T cells can complete division cycles in less than 6 h, representing a physiological extreme for somatic mammalian cells. Embryonic stem cells utilize specialized cell cycle control mechanisms, including subdued periodic expression, for rapid cell division cycles. CD8 T cell cycle control remains poorly understood. Here, we test whether CD8 T cells utilize embryonic mechanisms to promote rapid cell cycles. We comprehensively measure protein abundances in G1, S, and G2&M phases in three murine cell types: CD8 T cells, embryonic stem cells, and fibroblasts. We discover striking similarities between mESC and CD8 T cells. We demonstrate that CD8 T cells express Cyclin E1 and Emi1/Fbxo5 at high levels to promote S-phase entry. Interestingly, CD8 T cells and mESCs differ in the frequency of G2&M phase cells, the abundance of DNA replication origin licensing and initiation factors, and the abundance of APC/C substrates. Thus, somatic T cells have both unique and shared cell cycle control mechanisms to promote rapid cell cycles.

IntroductionFollowing antigenic stimulation, T cells switch from a catabolic metabolic state maintained by low levels of nutrient uptake to an anabolic metabolism that sustains the biosynthetic and energetic demands of clonal expansion, differentiation, and effector function. Much progress has been made in understanding the transcriptional and enzymatic regulation of activated T cell metabolism. However, less is understood of the role for regulators of anaplerosis and cataplerosis such as phospho-enol pyruvate carboxykinases (PEPCK) in T cells. MethodsAssessment of PEPCK expression in mouse T cells was performed. Pharmacological inhibitors were used to assess functional and metabolic roles for PEPCKs in T cell activation. ResultsWe show that mitochondrial PEPCK (PEPCK-M) is upregulated following T cell activation, while cytosolic PEPCK-C was not detected. The PEPCK inhibitors limited CD8+ T cell cytotoxic capacity and both CD4+ and CD8+ T cell inflammatory cytokine production. The suppression of T cell effector functions by PEPCK inhibitors was associated with decreased maximal mitochondrial respiration.DiscussionThese data suggest that PEPCKs act to modulate mitochondrial metabolism, supporting effector function in T cells.

Rapid proliferation and expansion of cytotoxic CD8 T lymphocytes is crucial for adaptive immunity against viral infection. CD8 T cell cycles complete cell division cycles in <6 hours, representing a physiological extreme for somatic mammalian cells. Embryonic stem cells also rapidly divide and have been shown to utilize specialized cell cycle control mechanisms that differ from somatic fibroblasts, including subdued periodicity of cyclin proteins. CD8 T cell cycle control remains poorly understood compared to embryonic and other somatic cell types. Here, we tested whether CD8 T cells utilize similar control mechanisms to embryonic stem cells to promote rapid cell cycles. We used mass spectrometry-based proteomics to comprehensively measure protein abundances in G1, S and G2&M phases somatic mouse CD8 T cells, mouse embryonic stem cells (mESC) and mouse fibroblasts (NIH3T3). We isolated cell cycle phases using PRIMMUS, thereby avoiding potential artefacts due to arrest-based synchronization. We discovered striking similarities between mESC and CD8 T cells. Similar to mESC, Cyclin E1 was expressed at high levels and constitutively across the cell cycle in CD8 T cells. Overall, cyclins and cell cycle regulated proteins were present in higher abundance in CD8 T cells and mESC as compared with NIH3T3 cells. Additionally, CD8 T cells express high levels of Emi1/Fbxo5 to promote S-phase entry. Interestingly, Emi1 deletion unexpectedly resulted in changes in markers of CD8 T cell phenotype, suggesting an association between cell cycle control and immune function. Thus, we show several adaptations of cell cycle control of embryonic stem cells are redeployed in somatic T cells to promote rapid cell cycles. ### Competing Interest Statement The authors have declared no competing interest.

Argonautes are ancient proteins with well-characterised functions in cell-autonomous gene regulation and genome defense but less clear roles in non-cell-autonomous processes. Extracellular Argonautes have been reported across plants, animals and protozoa yet their biochemical and functional properties remain elusive. Here we demonstrate that an extracellular Argonaute (exWAGO) released by the rodent-infective parasitic nematode Heligmosomoides bakeri is detectable inside mouse cells during the natural infection. We further show that exWAGO is released from H.bakeri in both vesicular and non-vesicular forms that have different resistances to proteolysis, different accessibilities to antibodies and associate with different subsets of secondary siRNAs. Using recombinant exWAGO protein we demonstrate that non-vesicular exWAGO is directly internalised by mouse cells in vitro and that immunisation of mice with exWAGO confers partial protection against subsequent H. bakeri infection and generates antibodies that block exWAGO uptake into cells. Finally, we show that properties of exWAGO are conserved across Clade V nematodes that infect humans and livestock. Together this work expands the context in which Argonautes function and illuminates an RNA-binding protein as a vaccine target for parasitic nematodes.

Following antigenic stimulation, T cells switch from a catabolic metabolic state maintained by low levels of nutrient uptake to an anabolic metabolism that sustains the biosynthetic and energetic demands of clonal expansion, differentiation and effector function. Much progress has been made in understanding the transcriptional and enzymatic regulation of activated T cell metabolism. However less is understood of the role for regulators of anaplerosis and cataplerosis such as phospho-enol pyruvate carboxykinases (PEPCK) in T cells. In the current work, we show that mitochondrial isoform PEPCK-M is upregulated following T cell activation whilst cytosolic PEPCK-C is not expressed. PEPCK inhibitors limited CD8+ T cell cytotoxic capacity and both CD4+ and CD8+ T cell inflammatory cytokine production. Suppression of T cell effector functions by PEPCK inhibitors was associated with decreased maximal mitochondrial respiration. These data suggest that PEPCK-M acts as a metabolic rheostat to enable optimal T cell activation. ### Competing Interest Statement The authors have declared no competing interest. Cancer Research UK, https://ror.org/054225q67, 23269

BACKGROUND:Adoptive cell therapy (ACT) is a promising strategy for treating cancer, yet it faces several challenges such as lack of long-term protection due to T cell exhaustion induced by chronic TCR stimulation in the tumor microenvironment. One benefit of ACT, however, is that it allows for cellular manipulations, such as deletion of the phosphotyrosine phosphatase non-receptor type 22 (PTPN22), which improves CD8+ T cell antitumor efficacy in ACT. We tested whether Ptpn22KO cytolytic T cells (CTLs) were also more effective than Ptpn22WT CTL in controlling tumors in scenarios that favor T cell exhaustion.METHODS:Tumor control by Ptpn22WT and Ptpn22KO CTL was assessed following adoptive transfer of low numbers of CTL to mice with subcutaneously implanted MC38 tumors. Tumor infiltrating lymphocytes were isolated for analysis of effector functions. An in vitro assay was established to compare CTL function in response to acute and chronic restimulation with antigen-pulsed tumor cells. The expression of effector and exhaustion-associated proteins by Ptpn22WT and Ptpn22KO T cells was followed over time in vitro and in vivo using the ID8 tumor model. Finally, the effect of PD-1 and TIM-3 blockade on Ptpn22KO CTL tumor control was assessed using monoclonal antibodies and CRISPR/Cas9-mediated knockout.RESULTS:Despite having improved effector function at the time of transfer, Ptpn22KO CTL became more exhausted than Ptpn22WT CTL, characterized by more rapid loss of effector functions, and earlier and higher expression of inhibitory receptors (IRs), particularly the terminal exhaustion marker TIM-3. TIM-3 expression, under the control of the transcription factor NFIL3, was induced by IL-2 signaling which was enhanced in Ptpn22KO cells. Antitumor responses of Ptpn22KO CTL were improved following PD-1 blockade in vivo, yet knockout or antibody-mediated blockade of TIM-3 did not improve but further impaired tumor control, indicating TIM-3 signaling itself did not drive the diminished function seen in Ptpn22KO CTL.CONCLUSIONS:This study questions whether TIM-3 plays a role as an IR and highlights that genetic manipulation of T cells for ACT needs to balance short-term augmented effector function against the risk of T cell exhaustion in order to achieve longer-term protection.

Abstract Background Following vaccination against SARS-CoV-2 (SC2), the human immune system produces neutralizing antibodies (NAb) that block the virus from entering host cells. These NAb levels are highly predictive of protection against SC2 infection and progression to severe disease [1]. Unfortunately, individual humoral responses vary and NAb titers decay over time, making measurement of NAb important in timing vaccine boosters, especially for at-risk individuals. Microneutralization assays (MNA) can be used to measure vaccine effectiveness by quantifying virus-specific NAb titers. However, these assays have long turnarounds, require a BSL3 lab, and have high inter-lab variability [2]. The Q-NAb™ test was developed to quantitatively measure NAb levels and correlate with MNA [3]. Q-NAb is available as both an ELISA for central laboratories, and a multiplexed POC centrifugal disc assay that measures NAb, anti-Nucleocapsid protein (NP), and anti-Spike antibodies. Methods The heart of the Q-NAb test is a recombinant fusion protein (FP) that blocks anti-RBD non-neutralizing antibodies (NNAb). FP consists of human ACE2 fused to the Wuhan-RBD of the SC2 Spike. Designed to conceal the the NAb binding epitopes of RBD, FP serves as a specific depleting agent for NNAb. The pretreated sample is then incubated with immobilized Wuhan-RBD to obtain a NAb readout. Both Q-NAb formats were calibrated to WHO international standard 20/136 using a set of 7 calibrators and correlated to MNA results. NAb levels were determined using >200 clinical samples collected 2–12 weeks post monovalent or bivalent vaccine booster and compared across age groups (<60, 60–70, 70–80, 80+ years). Results Both Q-NAb Disc and ELISA show correlation to each other (n = 167, WLS, R2 = 0.85) and are traceable to international standards. Q-NAb also correlated with MN50: ELISA (n = 137, Spearman's ρ = 0.88) and Disc (n = 208, Spearman's ρ = 0.91). After receiving a bivalent booster, no difference in median NAb levels across age groups was observed (Kruskal-Wallis, P = 0.3). While 99.4% of samples had a positive anti-Spike, using a NAb cutoff of 1000 IU/mL derived from the literature [2, 4–5], we found the percentage of individuals below that cutoff increased with age (9.8% for <60 years, 20.4% for 60–70 years, 19.5% for 70–80 years, and 34.1% for 80+ years). In addition, 32% of samples had an anti-NP level indicating recent infection. Conclusion The Q-NAb test, available in both central lab and POC formats, measures NAb levels, correlates to MNA, and is traceable to international standards. Q-NAb was used to assess humoral responses following vaccine boosters in a clinical sample set. While virtually all subjects had anti-Spike levels, the percentage of individuals falling below a NAb cutoff of 1000 IU/mL ranged from 9.8% to 34.1%, depending on age. Additionally, about one-third of patients had anti-NP activity, indicating NAb levels resulted from a combination of vaccination and infection. This data shows Q-NAb is an easily accessible and more accurate tool to identify a subset of at-risk patients that anti-Spike antibody tests alone do not discriminate.

Control of mRNA translation adjusts protein production rapidly and facilitates local cellular responses to environmental conditions. Traditionally initiation of translation is considered to be a major translational control point, however, control of peptide elongation is also important. Here we show that the function of the elongation factor, eIF5a, is regulated dynamically in naïve CD8+ T cells upon activation by post-translational modification, whereupon it facilitates translation of specific subsets of proteins. eIF5a is essential for long-term survival of effector CD8+ T cells and sequencing of nascent polypeptides indicates that the production of proteins which regulate proliferation and key effector functions, particularly the production of IFNγ and less acutely TNF production and cytotoxicity, is dependent on the presence of functional eIF5a. Control of translation in multiple immune cell lineages is required to co-ordinate immune responses and these data illustrate that translational elongation contributes to post-transcriptional regulons important for the control of inflammation.

RMRP encodes a non-coding RNA forming the core of the RNase MRP ribonucleoprotein complex. Mutations cause Cartilage Hair Hypoplasia (CHH), characterized by skeletal abnormalities and impaired T cell activation. Yeast RNase MRP cleaves a specific site in the pre-ribosomal RNA (pre-rRNA) during ribosome synthesis. CRISPR-mediated disruption of RMRP in human cells lines caused growth arrest, with pre-rRNA accumulation. Here, we analyzed disease-relevant primary cells, showing that mutations in RMRP impair mouse T cell activation and delay pre-rRNA processing. Patient-derived human fibroblasts with CHH-linked mutations showed similar pre-rRNA processing delay. Human cells engineered with the most common CHH mutation (70AG in RMRP) show specifically impaired pre-rRNA processing, resulting in reduced mature rRNA and a reduced ratio of cytosolic to mitochondrial ribosomes. Moreover, the 70AG mutation caused a reduction in intact RNase MRP complexes. Together, these results indicate that CHH is a ribosomopathy.

Adoptive T cell transfer has improved the treatment of cancer patients. However, treatment of solid tumors is still challenging and new strategies that optimize T cell function and response duration in the tumor could be beneficial additions to cancer therapy. In this study, we deleted the intracellular phosphatase PTPN22 and the endogenous TCR α chain from human PBMC-derived T cells using CRISPR/Cas9 and transduced them with TCRs specific for a defined antigen. Deletion of PTPN22 in human T cells increased the secretion of IFNγ and GM-CSF in multiple donors. The cells retained a polyfunctional cytokine expression after re-stimulation and greater numbers of PTPN22 KO T cells expressed inflammatory cytokines compared to unmutated control cells. PTPN22 KO T cells seemed to be more polyfunctional at low antigen concentrations. Additionally, we were able to show that that PTPN22 KO T cells were more effective in controlling tumor cell growth. This suggests that they might be more functional within the suppressive tumor microenvironment thereby overcoming the limitations of immunotherapy for solid tumors.

Lymphopenic insult has been shown to precipitate the initiation of autoimmune disease in murine models such as the Non-obese diabetic mouse. Similarly, in man lymphopenia induced by mAb therapy, for instance Alemtuzumab as treatment for Multiple Sclerosis, can precipitate development of secondary autoimmune disease in up to 30 % of patients. We asked whether an identified autoimmune susceptibility locus might increase the risk of developing autoimmunity in the context of mAb-induced lymphopenia in a mouse model. A single nucleotide polymorphism (SNP) in the gene encoding the tyrosine phosphatase PTPN22 (R620W) is associated with multiple human autoimmune diseases, and PTPN22 has been shown to modulate T cell responses, particularly to weak antigens. In keeping with this, PTPN22-deficient or PTPN22 R619W mutant murine T cells adoptively transferred into immunodeficient lymphopenic hosts showed a higher lymphopenia-induced proliferation rate than WT cells. We induced lymphopenia by treating wild-type or PTPN22 knock-out mice with T cell depleting antibodies and monitored reconstitution of the T cell pool. We found that PTPN22 deficient T cells acquired a more activated effector phenotype, with significantly more IFNγ producing cells. This resulted from expansion driven by self-peptide MHC, as it was evident when the contribution of IL-7 to lymphopenic expansion was blocked with IL-7R Ab. Interestingly, Foxp3+ Tregs were also considerably expanded in PTPN22-deficient and PTPN22 R619W mice, as was the frequency of both CD25+ and CD25- CD4 T cells that produce IL-10. Using bone marrow chimeric mice, we showed that PTPN22 influenced development of both regulatory and effector T cell functions in a cell-intrinsic manner. Overall the expansion of Tregs is likely to keep the expanded T effector populations in check and sparing Treg during therapeutic mAb depletion may be a useful strategy to prevent occurrence of secondary autoimmunity.

Adoptive T cell therapy (ACT) has been established as an efficacious methodology for the treatment of cancer. Identifying targets to enhance the antigen recognition, functional capacity, and longevity of T cells has the potential to broaden the applicability of these approaches in the clinic. We previously reported that targeting expression of phosphotyrosine phosphatase, nonreceptor type 22 (PTPN22) in effector CD8(+) T cells enhances the efficacy of ACT for tumor clearance in mice. In the current work, we demonstrate that, upon ACT, PTPN22-deficient effector CD8(+) T cells afforded greater protection against tumors expressing very low-affinity antigen but did not survive long term in vivo. Persistence of CD8(+) T cells following tumor clearance was improved by ACT of memory phenotype cells that have a distinct metabolic phenotype, as compared with effector T cells, Importantly, PTPN22-deficient T cells have comparable capacity to form long-lived memory cells in vivo but enhanced antitumor activity in vivo and effector responses ex vivo. These findings provide key insights into the regulation of effector and memory T cell responses in vivo and indicate that PTPN22 is a rational target to improve ACT for cancer.

A single nucleotide polymorphism, C1858T, in the gene encoding the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) results in one of the strongest genetic traits associated with autoimmune disease outside of the Major Histocompatibility Complex (MHC) genes. However, the consequences of this polymorphism, which introduces an arginine to tryptophan substitution at amino acid 620, for the function of PTPN22 protein is unclear and conflicting results have been obtained in human compared to mouse cells expressing this variant phosphatase. In mouse the variant appears to be a loss-of-function allele resembling a milder form of the null allele, while studies in human cells have reported it to be a gain-of-function mutation. To address whether the phosphatase has distinct functions in mouse vs. human T cells, we used CRISPR gene-editing to generate the first example of human PTPN22-KnockOut (KO) T cells. By comparing isogenic human T cells which express or lack PTPN22, we showed that PTPN22 KO T cells displayed enhanced expression of IL-2 and CD69 upon stimulation with cognate antigen. PTPN22 KO cells also showed increased Erk phosphorylation upon stimulation with weak antigen, but the difference was diminished in response to strong antigen, indicating that PTPN22 plays a more critical role in regulating weak-antigen responses. These data are in keeping with a role for PTPN22 in determining the threshold of stimulation required to activate T cells, a critical function of autoimmune pathogenesis. Our data indicate that PTPN22 has comparable functions in mouse and human T cells, and that the conflicting results in the literature regarding the impact of the point mutation are not due to differences in the activity of PTPN22 itself, but may be related to interactions with other proteins or splice variation.

In The Lancet Infectious Diseases, Catherine Ison and colleagues1Ison C Town K Obi C et al.on behalf of the GRASP collaborative groupDecreased susceptibility to cephalosporins among gonococci: data from the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) in England and Wales, 2007–2011.Lancet Infect Dis. 2013; (published online June 11.)http://dx.doi.org/10.1016/S1473-3099(13)70143-9PubMed Google Scholar describe recent dissemination (2007–11) of the penA mosaic gene in Neisseria gonorrhoeae in England and Wales, particularly among men who have sex with men. Their findings indicate a creeping resistance to cefixime—the former first-line treatment for gonorrhoea—and much slower development of ceftriaxone resistance, both with breakpoints of 0·25 mg/L. This decreased susceptibility has been reported previously in the USA,2Bolan GA Sparling PF Wasserheit JN The emerging threat of untreatable gonococcal infection.N Engl J Med. 2012; 366: 485-487Crossref PubMed Scopus (218) Google Scholar most typically in men who have sex with men and with similar relation to the mosaic penA resistance gene. Isolated cases of resistance to these drugs have also been recorded in France and Spain.3Cámara J Serra J Ayats J et al.Molecular characterization of two high-level ceftriaxone-resistant Neisseria gonorrhoeae isolates detected in Catalonia, Spain.J Antimicrob Chemother. 2012; 67: 1858-1860Crossref PubMed Scopus (267) Google Scholar In Japan, although cefixime was not used as standard treatment until the mid 2000s, a sporadic case of resistance was noted in a man in 2001.4Ito M Yasuda M Yokoi S et al.Remarkable increase in central Japan in 2001–2002 of Neisseria gonorrhoeae isolates with decreased susceptibility to penicillin, tetracycline, oral cephalosporins, and fluoroquinolones.Antimicrob Agents Chemother. 2001; 48: 3185-3187Crossref Scopus (90) Google Scholar However, in general, the prevalence of ceftriaxone resistance in southeast Asia is around 5%, but resistance is unrelated to the penA mosaic gene.5Olsen B Pham TL Golparian D Johansson E Tran HK Unemo M Antimicrobial susceptibility and genetic characteristics of Neisseria gonorrhoeae isolates from Vietnam, 2011.BMC Infect Dis. 2013; 13: 40Crossref PubMed Scopus (51) Google Scholar Preliminary work also shows that the common sequence type of N gonorrhoeae in England and Wales, G1407, is noted rarely in people infected in southeast Asia.5Olsen B Pham TL Golparian D Johansson E Tran HK Unemo M Antimicrobial susceptibility and genetic characteristics of Neisseria gonorrhoeae isolates from Vietnam, 2011.BMC Infect Dis. 2013; 13: 40Crossref PubMed Scopus (51) Google Scholar The appearance of cephalosporin resistance in men who have sex with men is no surprise; it parallels the resurgence of syphilis in Dublin, Ireland, where 83% of new cases arose in this group.6Muldoon E Mulcahy F Syphilis resurgence in Dublin, Ireland.Int J STD AIDS. 2011; 9: 493-497Crossref Scopus (25) Google Scholar These men have more sexual partners over a longer period than do their heterosexual counterparts, and diffuse sexual networks make contact-tracing less effective. Use of a molecular probe in men who have sex with men could help to control the gonorrhoea epidemic, with rapid detection of resistant strains of N gonorrhoeae and the mosaic penA gene and improved contact-tracing. Molecular probes are more sensitive for identification of gonococci compared with routine culture from non-urethral sites in men.7Bignall C Ison C FitzGerald M United Kingdom national guideline for gonorrhoea testing 2012.http://www.bashh.org/documents/4490.pdfGoogle Scholar The received wisdom of groups that monitor gonococcal antibiotic resistance—such as the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) in England and Wales—is that first-line treatment for gonorrhoea should be suspended whenever findings of sentinel resistance studies show that more than 5% of isolates become resistant. This threshold has been reached with cefixime: Ison and colleagues reported that 8·9% of isolates were resistant to cefixime between 2007 and 2011. A gradual increase in the minimum inhibitory concentration (MIC) of ceftriaxone was also noted during this period, although only nine of 6167 isolates were technically resistant. This creeping ceftriaxone resistance has led some to suggest administration of a double dose of ceftriaxone (500 mg intramuscularly) once only, possibly combined with another antimicrobial.7Bignall C Ison C FitzGerald M United Kingdom national guideline for gonorrhoea testing 2012.http://www.bashh.org/documents/4490.pdfGoogle Scholar Development of gradual resistance, unfortunately, is a fact of life. The only exception that comes to mind is the sensitivity of Treponema pallidum to penicillin, which has not altered since introduction of this antibiotic in the 1940s. Even here, however, a theoretical mechanism of resistance exists.8Cha JY Ishiwata A Mobishery S A novel beta-lactamase activity from a penicillin-binding protein of Treponema pallidum and why syphilis is still treatable with penicillin.J Biol Chem. 2004; 279: 14917-14921Crossref PubMed Scopus (39) Google Scholar What is more encouraging is the return of antibiotic sensitivity with disuse of an agent; thus, abandoned drugs can again be useful. If we look at the changing sensitivities of antibiotics for N gonorrhoeae,9Davies SC Fowler T Watson J Livermore DM Walker D Annual Report of the Chief Medical Officer: infection and the rise of antimicrobial resistance.Lancet. 2013; 381: 1606-1609Summary Full Text Full Text PDF PubMed Scopus (179) Google Scholar from 2000 to 2013, a slight decline was noted in ciprofloxacin resistance but a more substantial fall was seen for penicillin resistance. Ison and colleagues did not report ceftriaxone resistance in the last year of surveillance, perhaps indicating that a similar loss of resistance has happened. A new drug on the horizon is the fluoroketolide solithromycin, to which N gonorrhoeae is very sensitive in vitro, and this agent is currently in pneumonia trials.10Golparian D Fernandes P Ohnishi M Jensen JS Unemo M In vitro activity of the new fluoroketolide solithromycin (CEM-10) against a large collection of clinical Neisseria gonorrhoeae isolates and international reference strains including high-level antimicrobial resistance: potential treatment option for gonorrhoea?.Antimicrob Agents Chemother. 2012; 56: 2739-2742Crossref PubMed Scopus (83) Google Scholar Reintroduction of spectinomycin is another possibility, because gonococcal resistance to this drug is very unusual.11Unemo M Golparian D Skogen V et al.Neisseria gonorrhoeae strain with high-level resistance to spectinomycin due to a novel resistance mechanism (mutated ribosomal protein S5) verified in Norway.Antimicrob Agent Chemother. 2013; 57: 1057-1061Crossref PubMed Scopus (61) Google Scholar Most promising is combined use of antimicrobials, which in practice can delay the emergence of resistant microorganisms, contrary to the theory of Pena-Miller and colleagues.12Pena-Miller R Laehnemann D Jansen G et al.When the most potent combination of antibiotics selects for the greatest bacterial load: the smile-frown transition.PLoS Biol. 2013; 11: e1001540Crossref PubMed Scopus (147) Google Scholar Candidate antibiotics for coadministration with ceftriaxone are azithromycin, gentamicin, and doxycycline. The combination of azithromycin and gemifloxacin is also in phase 4 trials in the USA. Empirically, none of these antimicrobials should be given alone: the MIC of azithromycin13European Committee on Antimicrobial Susceptibility TestingBreakpoint tables for interpretation of MICs and zone diameters, version 1.3.http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_test_documents/EUCAST_breakpoints_v1.3_pdf.pdfDate: 2011Google Scholar is 2 mg/L (resistance is about 0·4%), the preliminary MIC of gentamicin14Brown LB Krysiak R Kamanga G et al.Neisseria gonorrhoeae antimicrobial susceptibility in Lilongwe, Malawi, 2007.Sex Transm Dis. 2010; 37: 169-172Crossref PubMed Scopus (88) Google Scholar is 4 mg/L (10% resistance), and the MIC of doxycycline (using tetracycline as a surrogate)13European Committee on Antimicrobial Susceptibility TestingBreakpoint tables for interpretation of MICs and zone diameters, version 1.3.http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_test_documents/EUCAST_breakpoints_v1.3_pdf.pdfDate: 2011Google Scholar is 2 mg/L (19% resistance). The combination of azithromycin and ceftriaxone is only additive and no synergistic action exists between them. Finally, rifamides might be a possible treatment for gonorrhoea, although when rifampicin is given alone, up to two-thirds of isolates show intermediate rifampicin resistance.15Sutrisna A Soebjakto O Wignall FS et al.Increasing resistance to ciprofloxacin and other antibiotics in Neisseria gonorrhoeae from East Java and Papua, Indonesia, in 2004: implications for treatment.Int J STD AIDS. 2006; 17: 810-812Crossref PubMed Scopus (22) Google Scholar How can we manage the changing face of gonorrhoea? 90 years ago, Thomson16Thomson D Gonorrhoea. Henry Frowde and Oxford University Press, London1923: 378Google Scholar regarded gonorrhoea as a common cause of blindness, spread by travellers but difficult (and impractical) to combat by simply preaching chastity. Modern epidemiology and antimicrobials have replaced this approach; nevertheless, successful use of drugs has to be monitored in view of evolving patterns of resistance. If the current recommended treatment regimen of combined azithromycin and ceftriaxone7Bignall C Ison C FitzGerald M United Kingdom national guideline for gonorrhoea testing 2012.http://www.bashh.org/documents/4490.pdfGoogle Scholar falters, the long-term solution could be development of a vaccine against gonorrhoea. We declare that we have no conflicts of interests. Decreased susceptibility to cephalosporins among gonococci: data from the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) in England and Wales, 2007–2011Guidance for treatment of gonorrhoea in England and Wales was changed in 2010 to prolong the use of cephalosporins. The decline in prevalence of isolates with decreased cefixime susceptibility cannot be attributed unequivocally to this change in prescribing practice; however, the association is striking. Full-Text PDF Decreased susceptibility to cephalosporins among gonococci? – Authors' replyWe read with interest the Correspondence about our Article on decreased susceptibility to cephalosporins among gonococci in England and Wales,1 and welcome the data shared from Australia, France, and Japan. We are encouraged to note that, in Australia, gonococcal isolates with decreased susceptibility to ceftriaxone fell from 2010 to 2011, similar to our findings for susceptibility to cefixime. In both instances, the fall was associated with increased dosage of ceftriaxone; albeit as monotherapy in Australia, whereas dual therapy (in combination with azithromycin) was used in the UK. Full-Text PDF Decreased susceptibility to cephalosporins among gonococci?We read with interest the Article by Catherine Ison and colleagues1 and the Comment by David Wright and Berge Azadian2 about decreased susceptibility to cephalosporins among clinical strains of Neisseria gonorrhoeae. The papers describe the association of the increase of such strains in men who had sex with men (MSM) and discuss strategies to treat gonorrhoea in the era of emergence and spread of cephalosporin resistance among gonococci. Full-Text PDF

Intragenic spacer (IGS, between 16S–23S genes) provides resolution among Lyme-borreliosis-associated spirochaetes and some relapsing fever Borrelia. When applied to East African relapsing fever borreliae, two and four types were found, respectively, among B. recurrentis and B. duttonii. Surprisingly, IGS typing was unable to discriminate between the tick- and louse-borne forms of disease, raising the possibility that these organisms are the same species. In order to resolve this question, further genes were sequenced to produce a multi-locus approach to determine whether these are indeed different species. Various housekeeping genes were selected from data deposited for B. hermsii (limited sequence information exists for either B. recurrentis or B. duttonii). Of selected targets, sufficient amplification was only produced using glpQ; thus, further genes were analysed including flaB, rrs rDNA, and p66 outer membrane protein. The fastidious nature of these organisms limited numbers of isolates available for full analysis. In contrast, the IGS typing was applied to a variety of patient's blood samples and arthropod vectors in addition to cultivable isolates, potentially introducing a bias to the results. Results highlighted the remarkable similarity between these B. recurrentis and B. duttonii, with only minor nucleotide differences. Collectively, these findings suggest a common ancestral lineage for these spirochaetes. These minor nucleotide differences of 2–10 nucleotides were able to differentiate both ‘species’. It is likely that these differences reflect an accumulation of adaptive changes through time and pressures resulting from different vector transmission, with these spirochaetes being subgroups of the same species. In contrast, the IGS sequence, being non-coding, is not under such selective pressure and thus probably reflects changes accumulated over time alone, without the functional constraints. Complete genomic sequence analysis is likely to illuminate greater insights into the taxonomic relationship between these microbes and elucidate the molecular basis of arthropod competence and pathogenicity among these spirochaetes.

Lyme disease is being increasingly diagnosed in the UK. The clinical picture, laboratory diagnosis and treatment of the disease are described and aspects of the pathology and epidemiology are highlighted.

AnaesthesiaVolume 32, Issue 9 p. 916-917 Free Access A dangerous lignocaine spray D.J. Wright, D.J. Wright Department of Anaesthetics, The Royal Infirmary, Edinburgh EH3 9 YWSearch for more papers by this author D.J. Wright, D.J. Wright Department of Anaesthetics, The Royal Infirmary, Edinburgh EH3 9 YWSearch for more papers by this author First published: October 1977 https://doi.org/10.1111/j.1365-2044.1977.tb10127.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 19 Harley, H.R.S., Laurence, K.M., Seal, R.M.E. & Steven, J.H. (1965) Fatal cerebral anoxia following the use of lignocaine spray for bronchoscopy–a case report. British Journal of Anaesthesia, 37, 61. Volume32, Issue9October 1977Pages 916-917 ReferencesRelatedInformation