Vaccination with meningococcal outer membrane vesicles carrying Borrelia OspA protects against experimental Lyme borreliosis.

Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines, as such a vaccine has been proven to be successful in the past. The expression of recombinant antigens in meningococcal Outer Membrane Vesicles (OMVs), with the OMV functioning both as adjuvant and delivery vehicle, greatly enhances their potential. Immunization studies in mice have shown that OMV-based vaccines can protect against various pathogens and an OMV-based meningococcal vaccine is approved and available for human use. Because of its surface localization in Borrelia and the detailed knowledge regarding its immunogenicity and structure, OspA was chosen as a suitable lipoprotein to be tested as an OMV-based vaccine against Lyme borreliosis. We have previously shown that the OMV-OspA vaccine was immunogenic in mice and here we assessed the efficacy of OMV-OspA. We generated a second-generation OMV-OspA vaccine and vaccinated C3H/HeN mice with (EDTA extracted) meningococcal OMVs expressing OspA from B. burgdorferi strain B31. The adjuvant effect of empty OMVs on recombinant OspA was tested as well. We subsequently challenged mice with a subcutaneous injection of B. burgdorferi. Average antibody end-point titers against the OspA-OMV construct were high, although lower compared to the antibodies raised against recombinant OspA. Interestingly, antibody titers between recombinant OspA adjuvanted with aluminum hydroxide and recombinant OspA with OMV as adjuvant were comparable. Finally, qPCR and culture data show that both the OspA-OMV and the vaccine based on recombinant OspA with OMV as adjuvant provided significant, yet partial protection, against Borrelia infection. OMV-based vaccines using Borrelia (lipo)proteins are an easy and feasible vaccination method protecting against B. burgdorferi infection and could be a promising strategy in humans.

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

This corrects the article DOI: 10.1038/mi.2017.81.

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

Mucosal immunity is often required for protection against respiratory pathogens but the underlying cellular and molecular mechanisms of induction remain poorly understood. Here, systems vaccinology was used to identify immune signatures after pulmonary or subcutaneous immunization of mice with pertussis outer membrane vesicles. Pulmonary immunization led to improved protection, exclusively induced mucosal immunoglobulin A (IgA) and T helper type 17 (Th17) responses, and in addition evoked elevated systemic immunoglobulin G (IgG) antibody levels, IgG-producing plasma cells, memory B cells, and Th17 cells. These adaptive responses were preceded by unique local expression of genes of the innate immune response related to Th17 (e.g., Rorc) and IgA responses (e.g., Pigr) in addition to local and systemic secretion of Th1/Th17-promoting cytokines. This comprehensive systems approach identifies the effect of the administration route on the development of mucosal immunity, its importance in protection against Bordetella pertussis, and reveals potential molecular correlates of vaccine immunity to this reemerging pathogen.

Challenges in the Management of Breast Cancer in a Low Resource Setting in South East Asia.

BACKGROUND: Breast cancer is the second most common cancer in women in Cambodia, a low income country in SouthEast Asia. The Sihanouk Hospital Centre of Hope (SHCH) is a charity hospital set up by an international nongovernmental organisation, HOPE Worldwide. In 2008, SHCH partnered with AmeriCares, a global health organisation to set up and deliver a breast cancer programme to provide education, diagnosis and treatment for women with breast cancer. The objective of this study is to characterise the presentation, diagnosis, treatment and outcomes of women treated under this program. MATERIALS AND METHODS: A total of 215 women newly diagnosed with breast cancer from 1 March 2008 until 31 March 2011 were studied. Age at diagnosis, tumour size, histological type, tumour grade, ER, lymph node involvement, treatment modalities (surgery, radiotherapy, chemotherapy, hormone therapy) were recorded. Data on mortality at 3 years were obtained whenever possible. RESULTS: The median age was 47 years old. Some 77.8% were diagnosed with stage 3 and 4 lesions, and 78.5% underwent mastectomy, of which 28.4% the intent was palliative. Of those whose ER status were known, only 48.3% were ER positive. Only 6 patients could afford chemotherapy while only 1 patient had radiotherapy. Hormone therapy was provided free for those who were ER positive. The overall survival rate at 3 years was 39.1%. CONCLUSIONS: Breast cancer presents at a late stage, and because treatment is suboptimal, survival is poor in Cambodia. A more aggressive approach to early detection and treatment needs to be developed to improve outcome from this potentially curable disease.

Trichinella spiralis-secreted products modulate DC functionality and expand regulatory T cells in vitro.

Helminths and their products can suppress the host immune response which may benefit parasite survival. Trichinella spiralis can establish chronic infections in a wide range of mammalian hosts including humans and mice. Here, we aim at studying the effect of T. spiralis muscle larvae excretory/secretory products (TspES) on the functionality of DC and T cell activation. We found that TspES suppress in vitro DC maturation induced by both S- and R-form lipopolysaccharide(LPS) from enterobacteria. Using different toll-like receptor (TLR) agonists, we show that the suppressive effect of TspES on DC maturation is restricted to TLR4. These helminth products also interfere with the expression of several genes related to the TLR-mediated signal transduction pathways. To investigate the effect of TspES on T cell activation, we used splenocytes derived from OVA-TCR transgenic D011.10 that were incubated with OVA and TspES-pulsed DC. Results indicate that the presence of TspES resulted in the expansion of CD4(+) CD25(+) Foxp3+ T cells. These regulatory T (Treg) cells were shown to have suppressive activity and to produce TGF-ß. Together these results suggest that T. spiralis secretion products can suppress DC maturation and induce the expansion of functional Treg cells in vitro.

Abundancia y distribución de Aedes aegypti (Diptera: Culicidae) y dispersión del dengue en Guasave Sinaloa, México

Abundance and distribution of Aedes aegypti (Diptera: Culicidae), and dengue dispersion in Guasave Sinaloa, México. Dengue is an important disease that affects humans, and is transmitted by A. aegypti. During 2006, a total of 477 cases of hemorrhagic dengue, and 1 510 of classic dengue were recorded in Sinaloa. Due to this high impact, a study on insect abundance and distribution, as well as their relationship with dengue dispersion, was carried out from April 2008 to March 2009 in Guasave, Sinaloa. The study included a total of six sectors in the city, considering 16 colonies; besides, 96 traps were distributed in these sectors to monitor the vector population density and female number per trap. The adult density index (ADI) and traps positive index (TPI) were calculated, and data were used to obtain the monthly dengue dispersion maps. The highest abundance of female (137 and 139) was found in July and August. In August and September the sectors 2, 4 and 6 showed the highest ADI values 2.44, 3.35 and 2.290, while TPI values were 56.25, 58.82 and 61.54, with the highest epidemiology dengue dispersion. The Pearson correlation (p<0.05) showed better values with the precipitation (r=0.80) than mean temperature (r=0.76) with zero lag months; while the cases of classic dengue and hemorrhagic dengue (CD/HD) is mostly related with the precipitation (r=0.98), and minimum temperature (r=0.79), with two lag months. In conclusion, the ADI and TPI index, as well as the maps obtained, allowed us to know the location of epidemiologic dengue risk areas. This information can be used to develop better control measures biological and chemical for the mosquito, in this location. Rev. Biol. Trop. 59 (4): 1609-1619. Epub 2011 December 01.

El dengue es una enfermedad viral en humanos transmitida por el mosquito A. aegypti (L), principal vector en América. En el 2006 en Sinaloa México se presentaron 477 casos de dengue hemorrágico y 1 510 de dengue clásico, el estado presentó el mayor número de casos de dengue hemorrágico del país, debido a esto se realizó un estudio sobre la abundancia y distribución del mosquito de abril 2008 a agosto 2009 en Guasave Sinaloa, México. La Ciudad se dividió en seis sectores, en la cual se consideraron 16 colonias con el mayor número de casas por sector; se colocaron 96 trampas para monitorear la densidad de población del insecto y el número de hembras por trampa, esto datos sirvieron para calcular los índices de densidad de adultos (IDA) e índice de trampas positivas (ITP) por sector, los cuales junto con los casos de dengue permitieron elaboraron mapas de dispersión de la enfermedad. En julio y agosto se registró la abundancia más alta de hembras (137 y 139). En agosto y septiembre los sectores dos, cuatro y seis presentaron valores de IDA de 2.44, 3.35 y 2.29, por lo que fueron de un alto riesgo epidemiológico, mientras que en estos mismos sectores se encontraron los valores más altos de ITP (56.25, 58.82 y 61.54). Los valores de mayor asociación de las correlaciones entre la abundancia poblacional de hembras se obtuvo con la precipitación (r=0.80) y con la temperatura promedio (r=0.76), a cero meses de retraso. La correlación entre el DC/DH con la precipitación fue más alta (r=0.98) que con la temperatura mínima (r=0.79); ambas con dos meses de retraso. En este estudio se demostró que los mapas mensuales de dispersión epidemiológica sirven como una medida del riesgo de la enfermedad en Guasave, Sinaloa.

Suppression of dendritic cell maturation by Trichinella spiralis excretory/secretory products.

Evidence from experimental studies indicates that during chronic infections with certain helminth species a regulatory network is induced that can down-modulate not only parasite-induced inflammation but also reduce other immunopathologies such as allergies and autoimmune diseases. The mechanisms however, and the molecules involved in this immunomodulation are unknown. Here, we focus on the effect of Trichinella spiralis excretory/secretory antigens (TspES) on the innate immune response by studying the effect of TspES on DC maturation in vitro. Bone marrow-derived DC from BALB/c mice were incubated with TspES either alone or in combination with LPS derived from two different bacteria. As indicators of DC maturation, the cytokine production (IL-1alpha, IL-6, IL-10, IL-12p70 and TNF-alpha) and the expression of various surface molecules (MHC-II, CD40, CD80 and CD86) were measured. Results indicate that while TspES alone did not change the expression of the different surface molecules or the cytokine production, it completely inhibited DC maturation induced by Escherichia coli LPS (E. coli LPS). In contrast, DC maturation induced by LPS from another bacterium, Neisseria meningitidis, was not affected by TspES. These results were confirmed using TLR4/MD2/CD14 transfected HEK 293 cells. In conclusion, T. spiralis ES antigens lead to suppression of DC maturation but this effect depends on the type of LPS used to activate these cells.

Incorporation of LpxL1, a detoxified lipopolysaccharide adjuvant, in influenza H5N1 virosomes increases vaccine immunogenicity.

The increasing number of human influenza H5N1 infections accentuates the need for the development of H5N1 vaccine candidates to prevent a potential influenza pandemic. The use of adjuvants in such vaccines can contribute significantly to antigen dose-sparing. In this study, we evaluated the capacity of the non-toxic Neisseria meningitidis lipopolysaccharide analog LpxL1 to function as an adjuvant for an influenza H5N1 virosomal vaccine. Inactivated influenza H5N1 virus (NIBRG-14) was used to construct virosomes (reconstituted virus envelopes) with LpxL1 incorporated in the virosomal membrane thus combining the influenza hemagglutinin (HA) antigen and the adjuvant in the same particle. Mice were immunized in a one- or two-dose immunization regimen with H5N1 virosomes with or without incorporated LpxL1. After a single immunization, H5N1 virosomes with incorporated LpxL1 induced significantly enhanced H5N1-specific total IgG titers as compared to non-adjuvanted virosomes but hemagglutination inhibition (HI) titers remained low. In the two-dose immunization regimen, LpxL1-modified H5N1 virosomes induced HI titers above 40 which were significantly higher than those obtained with non-adjuvanted virosomes. Incorporation of LpxL1 had little effect on virosome-induced IgG1 levels, but significantly increased IgG2a levels in both the one- and two-dose immunization regimen. Compared to non-adjuvanted virosomes, LpxL1-modified virosomes induced similar numbers of IFNgamma-producing T cells but decreased numbers of IL-4-producing T cells irrespective of the number of immunizations. We conclude that LpxL1 incorporated in H5N1 influenza virosomes has the capacity to function as a potent adjuvant particularly stimulating Th1-type immune reactions.

[Cord blood: from bench to bedside]. / Le sang de cordon: du laboratoire au lit du malade.

Since 1974, umbilical cord blood (CB) has been shown to contain haematopoietic stem cells similar to stem cells from the bone marrow. In 1988, E. Gluckman and her colleagues performed - successfully - the first familial CB transplantation and cured a 5 years old child suffering from Fanconi's anemia. Rapidly, CB banks were organised throughout in the world and thanks to this novel source of haematopoietic stem cells, we can now find a donor for 75 % of the patients requiring a "bone marrow" transplantation. The major benefit of CB as a source of hematopoietic stem cells is its easy access. CB also allows a more significant degree of HLA incompatibility and thus offers an opportunity of transplantation to ethnic minorities for whom no HLA identical donors are available. However, several studies have shown that the number of cells harvested in a CB was closely correlated with the engraftment post transplantation and today, a minimum of 3.7 x 10(7) mononucleated cells/kg is recommended. This required amount of cells is not always reached due to the small volume often harvested from a CB. Therefore, to apply CB transplantations to adults, different approaches are currently being investigated : coinfusion of haploidentical cells, mesenchymal cells, a second CB, or the addition of CB expanded ex-vivo. Among these approaches, double CB transplantation seems nowadays the most promising alternative and ongoing studies should soon inform us whether the duration of aplasia will be improved.

The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium-host interaction.

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.

High-level endothelial E-selectin (CD62E) cell adhesion molecule expression by a lipopolysaccharide-deficient strain of Neisseria meningitidis despite poor activation of NF-kappaB transcription factor.

Binding of host inflammatory cells to the endothelium is a critical contributor to the vascular damage characteristic of severe meningococcal disease and is regulated by endothelial cell adhesion molecules such as ICAM-1, VCAM-1 and CD62E. Intact meningococci induce far higher levels of CD62E than lipopolysaccharide (LPS) alone, whereas LPS is at least as potent as meningococci at inducing both VCAM-1 and ICAM-1 expression. This suggests that meningococci possess additional factors other than LPS present in whole bacteria that result in differential adhesion molecule expression. To investigate this possibility, we studied the capacity of an LPS-deficient isogenic strain of serogroup B Neisseria meningitidis H44/76 (lpxA-) to induce endothelial cell adhesion molecule expression and translocation of the transcription factor NF-kappaB, and compared it to both parent and unencapsulated strains of both B1940 and H44/76 and purified LPS. Although the LPS-deficient isogenic mutant of strain H44/76 was found to be a poor inducer of NF-kappaB, it induced higher levels of CD62E expression than LPS alone. These data provide evidence that intact meningococci induce a range of signals in the endothelium that are distinct from those seen with purified LPS alone and that they occur in a LPS-dependent and LPS-independent manner. These signals may explain the potent effects of N. meningitidis on CD62E expression on vascular endothelium and provide a basis for the complex endothelial dysregulation seen in meningococcal sepsis.

Functional activity of antibodies against the recombinant OpaJ protein from Neisseria meningitidis.

The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

Phase variation in meningococcal lipooligosaccharide biosynthesis genes.

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or lgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host.

Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant.

In the pathogen Neisseria meningitidis, a completely lipopolysaccharide (LPS)-deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis. To study how outer membrane structure and biogenesis are affected by the absence of this normally major component, inner and outer membranes were separated and their composition analysed. The expression and assembly of integral outer membrane proteins appeared largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins was greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards phosphatidylethanolamine and phosphatidylglycerol species containing mostly shorter chain, saturated fatty acids, one of which was unique to the LPS-deficient outer membrane. The presence of the capsular polysaccharide turned out to be essential for viability without LPS, as demonstrated by using a strain in which LPS biosynthesis could be switched on or off through a tac promoter-controlled lpxA gene. Taken together, these results can help to explain why meningococci have the unique ability to survive without LPS.

Phylogenetic relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA.

The ITS1, ITS2, and 5.8S gene sequences of nuclear ribosomal DNA from 40 taxa of the family Heteroderidae (including the genera Afenestrata, Cactodera, Heterodera, Globodera, Punctodera, Meloidodera, Cryphodera, and Thecavermiculatus) were sequenced and analyzed. The ITS regions displayed high levels of sequence divergence within Heteroderinae and compared to outgroup taxa. Unlike recent findings in root knot nematodes, ITS sequence polymorphism does not appear to complicate phylogenetic analysis of cyst nematodes. Phylogenetic analyses with maximum-parsimony, minimum-evolution, and maximum-likelihood methods were performed with a range of computer alignments, including elision and culled alignments. All multiple alignments and phylogenetic methods yielded similar basic structure for phylogenetic relationships of Heteroderidae. The cyst-forming nematodes are represented by six main clades corresponding to morphological characters and host specialization, with certain clades assuming different positions depending on alignment procedure and/or method of phylogenetic inference. Hypotheses of monophyly of Punctoderinae and Heteroderinae are, respectively, strongly and moderately supported by the ITS data across most alignments. Close relationships were revealed between the Avenae and the Sacchari groups and between the Humuli group and the species H. salixophila within Heteroderinae. The Goettingiana group occupies a basal position within this subfamily. The validity of the genera Afenestrata and Bidera was tested and is discussed based on molecular data. We conclude that ITS sequence data are appropriate for studies of relationships within the different species groups and less so for recovery of more ancient speciations within Heteroderidae.

Modification of lipid A biosynthesis in Neisseria meningitidis lpxL mutants: influence on lipopolysaccharide structure, toxicity, and adjuvant activity.

Two genes homologous to lpxL and lpxM from Escherichia coli and other gram-negative bacteria, which are involved in lipid A acyloxyacylation, were identified in Neisseria meningitidis strain H44/76 and insertionally inactivated. Analysis by tandem mass spectrometry showed that one of the resulting mutants, termed lpxL1, makes lipopolysaccharide (LPS) with penta- instead of hexa-acylated lipid A, in which the secondary lauroyl chain is specifically missing from the nonreducing end of the GlcN disaccharide. Insertional inactivation of the other (lpxL2) gene was not possible in wild-type strain H44/76 expressing full-length immunotype L3 lipopolysaccharide (LPS) but could be readily achieved in a galE mutant expressing a truncated oligosaccharide chain. Structural analysis of lpxL2 mutant lipid A showed a major tetra-acylated species lacking both secondary lauroyl chains and a minor penta-acylated species. The lpxL1 mutant LPS has retained adjuvant activity similar to wild-type meningococcal LPS when used for immunization of mice in combination with LPS-deficient outer membrane complexes from N. meningitidis but has reduced toxicity as measured in a tumor necrosis factor alpha induction assay with whole bacteria. In contrast, both adjuvant activity and toxicity of the lpxL2 mutant LPS are strongly reduced. As the combination of reduced toxicity and retained adjuvant activity has not been reported before for either lpxL or lpxM mutants from other bacterial species, our results demonstrate that modification of meningococcal lipid A biosynthesis can lead to novel LPS species more suitable for inclusion in human vaccines.

Defining the optimal surgeon experience for breast cancer sentinel lymph node biopsy: a model for implementation of new surgical techniques.

OBJECTIVE: To determine the optimal experience required to minimize the false-negative rate of sentinel lymph node (SLN) biopsy for breast cancer. SUMMARY BACKGROUND DATA: Before abandoning routine axillary dissection in favor of SLN biopsy for breast cancer, each surgeon and institution must document acceptable SLN identification and false-negative rates. Although some studies have examined the impact of individual surgeon experience on the SLN identification rate, minimal data exist to determine the optimal experience required to minimize the more crucial false-negative rate. METHODS: Analysis was performed of a large prospective multiinstitutional study involving 226 surgeons. SLN biopsy was performed using blue dye, radioactive colloid, or both. SLN biopsy was performed with completion axillary LN dissection in all patients. The impact of surgeon experience on the SLN identification and false-negative rates was examined. Logistic regression analysis was performed to evaluate independent factors in addition to surgeon experience associated with these outcomes. RESULTS: A total of 2,148 patients were enrolled in the study. Improvement in the SLN identification and false-negative rates was found after 20 cases had been performed. Multivariate analysis revealed that patient age, nonpalpable tumors, and injection of blue dye alone for SLN biopsy were independently associated with decreased SLN identification rates, whereas upper outer quadrant tumor location was the only factor associated with an increased false-negative rate. CONCLUSIONS: Surgeons should perform at least 20 SLN cases with acceptable results before abandoning routine axillary dissection. This study provides a model for surgeon training and experience that may be applicable to the implementation of other new surgical technologies.

Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response.

To determine the relative contribution of lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis to the pathogenesis of meningococcal sepsis, this study quantitatively compared cytokine induction by isolated LPS, wild-type serogroup B meningococci (strain H44/76), and LPS-deficient mutant meningococci (strain H44/76[pLAK33]). Stimulation of human peripheral-blood mononuclear cells with wild-type and LPS-deficient meningococci showed that non-LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production and virtually all interferon (IFN)-gamma production. Based on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LPS in proteinase K-treated lysates of N. meningitidis H44/76, a quantitative comparison was made between the cytokine-inducing capacity of isolated and purified LPS and LPS-containing meningococci. At concentrations of >10(7) bacteria/mL, intact bacteria were more potent cytokine inductors than equivalent amounts of isolated LPS, and cytokine induction by non-LPS components was additive to that by LPS. Experiments with mice showed that non-LPS components of meningococci were able to induce cytokine production and mortality. The principal conclusion is that non-LPS parts of N. meningitidis may play a role in the pathogenesis of meningococcal sepsis by inducing substantial TNF-alpha, IL-1beta, and IFN-gamma production.

Accuracy of sentinel lymph node biopsy for patients with T2 and T3 breast cancers.

Although numerous studies have demonstrated that sentinel lymph node (SLN) biopsy can accurately determine the axillary nodal status for early breast cancer some studies have suggested that SLN biopsy may be less reliable for tumors >2 cm in size. This analysis was performed to determine whether tumor size affects the accuracy of SLN biopsy. The University of Louisville Breast Cancer Sentinel Lymph Node Study is a prospective multi-institutional study involving 226 surgeons. The study was approved by the Institutional Review Board of each institution, and informed consent was obtained from all patients. Patients with clinical stage T1-2 N0 breast cancer were eligible for the study. Some patients with T3 tumors were included because they were clinically staged as T2 but on final pathology were found to have tumors >5 cm. This analysis includes 2148 patients who were enrolled from August 1997 through October 2000. All patients underwent SLN biopsy using a combination of radioactive colloid and blue dye injection followed by completion Level I/II axillary dissection. Statistical comparison was performed by chi-square analysis. The SLN identification rate, false negative rate, and overall accuracy of SLN biopsy were not significantly different among tumor stages T1, T2, and T3. We conclude that SLN biopsy is no less accurate for T2-3 breast cancers compared with T1 tumors.

Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria.

Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain, lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxA strains induced production of tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1alpha, IL-6, and TNF-alpha production and induced no detectable IL-12. Addition of exogenous LPS to the lpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.