Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCE: Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophil influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-NANA) scavenged from the host using LOS sialyltransferase (Lst), since Gc cannot make its own sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea.

Comparison of lipooligosaccharides from human challenge strains of Neisseria gonorrhoeae.

The alarming rise of antibiotic resistance and the emergence of new vaccine technologies have increased the focus on vaccination to control gonorrhea. Neisseria gonorrhoeae strains FA1090 and MS11 have been used in challenge studies in human males. We used negative-ion MALDI-TOF MS to profile intact lipooligosaccharide (LOS) from strains MS11mkA, MS11mkC, FA1090 A23a, and FA1090 1-81-S2. The MS11mkC and 1-81-S2 variants were isolated from male volunteers infected with MS11mkA and A23a, respectively. LOS profiles were obtained after purification using the classical phenol water extraction method and by microwave-enhanced enzymatic digestion, which is more amenable for small-scale work. Despite detecting some differences in the LOS profiles, the same major species were observed, indicating that microwave-enhanced enzymatic digestion is appropriate for MS studies. The compositions determined for MS11mkA and mkC LOS were consistent with previous reports. FA1090 is strongly recognized by mAb 2C7, an antibody-binding LOS with both α- and ß-chains if the latter is a lactosyl group. The spectra of the A23a and 1-81-S2 FA1090 LOS were similar to each other and consistent with the expression of α-chain lacto-N-neotetraose and ß-chain lactosyl moieties that can both be acceptor sites for sialic acid substitution. 1-81-S2 LOS was analyzed after culture with and without media supplemented with cytidine-5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which N. gonorrhoeae needs to sialylate its LOS. LOS sialylation reduces the infectivity of gonococci in men, although it induces serum resistance in serum-sensitive strains and reduces killing by neutrophils and antimicrobial peptides. The infectivity of FA1090 in men is much lower than that of MS11mkC, but the reason for this difference is unclear. Interestingly, some peaks in the spectra of 1-81-S2 LOS after bacterial culture with CMP-Neu5Ac were consistent with disialylation of the LOS, which could be relevant to the reduced infectivity of FA1090 in men and could have implications regarding the phase variation of the LOS and the natural history of infection.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia.

Societal attitudes influence performance on the Cognistat Judgment subtest.

The Cognistat is a widely used screening instrument for assessing cognitive functioning. However, the manualized scoring guidelines for one of the questions in the Judgment subtest appear insensitive to responses when examinees answer in light of current societal attitudes. Penalization of examinees is likely to lead to an underestimation of functional ability and a decrease in test validity. Suggestions for correcting this issue are presented.

Assessment of complications of EUS-guided fine-needle aspiration.

BACKGROUND: EUS-guided fine-needle aspiration (EUS-FNA) permits both morphologic and cytologic analysis of lesions within or adjacent to the GI tract. Despite increasing use of this technique, the safety and overall complication rates remain poorly defined. METHODS: During a period of 20 months, 322 consecutive patients underwent EUS-FNA in 2 centers. All procedures were performed with the patients under general anesthesia. All complications (including local complications resulting from endoscopy/aspiration or clinical complications after the procedure) were evaluated. Potential risk factors for the development of complications were also analyzed including site and nature of the lesion, presence of portal hypertension, and number of needle passes. RESULTS: A total of 345 lesions were aspirated in 322 patients. EUS-FNA involved the pancreas in 248 cases. Pancreatic lesions included solid (134) and cystic (114) types, which required a mean of 2.5 and 1.4 needle passes, respectively. Complications were observed in 4 (1.2%) patients after aspiration of pancreatic cystic lesions (acute pancreatitis, n = 3; aspiration pneumonia, n = 1) and all cases of pancreatitis resulted from FNA of lesions in the head/uncinate process. No complications resulted from FNA of solid pancreatic lesions. Complications were not observed after FNA of lymph nodes (n = 62) and one case of aspiration pneumonia was observed after FNA of a stromal tumor. EUS-FNA was performed without complication in 16 patients (5%) with portal hypertension. The number of needle passes was not predictive of complications. CONCLUSIONS: Because the overall risk of complications from EUS-FNA was relatively low (1.6%) with no severe or fatal incidents and although the risk appears slightly higher than that for standard EUS alone, the safety of EUS-FNA appears acceptable based on this analysis from an experienced center.

[Radiological aspects of hepatic tuberculoma. 3 cases]. / Aspects radiologiques du tuberculome hépatique. Trois cas.

Localized macronodular tuberculosis of the liver is rare. Tuberculous involvement of the liver is usually a diffuse process. We report 3 cases in which clinical and imaging features suggested the diagnosis of macronodular hepatic tuberculoma which was pathologically confirmed by percutaneous biopsy. In the first case, abdominal CT-scan showed a noncalcified hypodense nodular lesion in segment IV of the liver with an enhancing peripheral rim. In the second case, ultrasound showed 4 homogenous hypoechoic hepatic nodules. In the third case, a nodule was incidentally identified in segment VII of the liver. The lesion was hypoechoic with ultrasound, hypodense without enhancement on CT-scan, and hyperintense on both T1- and T2-weighted MR imaging. After percutaneous biopsy, pathologic examination showed peripheral granulomous lesions in all patients with central caseous necrosis consistent with tuberculosis in two patients despite a negative for Zielh stain. Specific M. tuberculosis culture was positive in the first patient, negative in the second patient, and was not performed in the third patient. Appropriate treatment resulted in disappearance of hepatic lesions on follow-up imaging examinations. These 3 cases show that clinical and imaging features of hepatic tuberculoma are not specific and that percutaneous biopsy of lesions provides a definite diagnosis.