Four Immune Modulating Genes in Primary Melanoma That Predict Metastatic Potential.

INTRODUCTION: Histologic characteristics cannot adequately predict which patients are at risk of developing metastatic disease after excision of primary cutaneous melanoma. The aim of this study was to identify immunomodulatory genes in primary tumors associated with development of distant metastases. MATERIALS AND METHODS: Thirty-seven patients with primary melanoma underwent surgical excision. RNA was extracted from the primary tumor specimens. cDNA was synthesized and used with Human Gene Expression microarray. Differential expression of 74 immunomodulatory genes was compared between patients who developed distant metastases and those who did not. RESULTS: Six of 37 patients developed distant metastases during the time of the study. Differential expression of microarray data showed upregulation of four immunomodulatory genes in this group. These four genes-c-CBL, CD276, CXCL1, and CXCL2-were all significantly overexpressed in the metastatic group with differential expression fold change of 1.15 (P = 0.01), 1.16 (P = 0.04), 2.51 (P < 0.001), and 1.68 (P < 0.02), respectively. CXCL1 had particularly high predictive value with an area under the curve of 0.80. Multivariate analysis showed only expression of CXCL1 (P = 0.01) remains predictive of distant metastases in melanoma patients. This result was confirmed using quantitative real-time polymerase chain reaction. CONCLUSIONS: CXCL1, CXCL2, c-CBL, and CD276 are immunomodulatory genes present in primary melanoma that are strongly associated with development of metastatic disease. Identification of their presence, particularly CXCL1, in the primary tumor could be used as a predictor of future risk of metastatic disease and thereby to identify patients who might benefit early from immunotherapy.

Real-Time PCR Reveals Rapid Dissemination of Leptospira interrogans after Intraperitoneal and Conjunctival Inoculation of Hamsters.

The pathogen Leptospira interrogans is a highly motile spirochete that causes acute and fulminant infections in humans and other accidental hosts. Hematogenous dissemination is important for infection by the pathogen but remains poorly understood because few animal model studies have used sensitive tools to quantify the bacteria. We evaluated the kinetics of leptospiral infection in Golden Syrian hamsters by a sensitive quantitative real-time PCR (TaqMan) with lipl32 as the target gene. The dissemination and bacterial burden were measured after intraperitoneal infection with a high dose (10(8)) or low dose (2.5 × 10(2)) of leptospires. We also examined the conjunctival challenge route to mimic the natural history of infection. Quantification of leptospires in perfused animals revealed that pathogens were detected in all organs of intraperitoneally infected hamsters, including the eye and brain, within 1 h after inoculation of 10(8) virulent L. interrogans bacteria. Peaks of 10(5) to 10(8) leptospires per gram or per milliliter were achieved in blood and all tissues between day 4 and day 8 after intraperitoneal inoculation of high- and low-dose challenges, respectively, coinciding with macroscopic and histological changes. The conjunctival route resulted in a delay in the time to peak organ burden in comparison to intraperitoneal infection, indicating that although infection could be established, penetration efficiency was low across this epithelial barrier. Surprisingly, infection with a large inoculum of high-passage-number attenuated L. interrogans strains resulted in dissemination to all organs in the first 4 days postinfection, albeit with a lower burden, followed by clearance from the blood and organs 7 days postinfection and survival of all animals. These results demonstrate that leptospiral dissemination and tissue invasion occur. In contrast, development of a critical level of tissue burden and pathology are dependent on the virulence of the infecting strain.

Oral immunization with Escherichia coli expressing a lipidated form of LigA protects hamsters against challenge with Leptospira interrogans serovar Copenhageni.

Leptospirosis is a potentially fatal zoonosis transmitted by reservoir host animals that harbor leptospires in their renal tubules and shed the bacteria in their urine. Leptospira interrogans serovar Copenhageni transmitted from Rattus norvegicus to humans is the most prevalent cause of urban leptospirosis. We examined L. interrogans LigA, domains 7 to 13 (LigA7-13), as an oral vaccine delivered by Escherichia coli as a lipidated, membrane-associated protein. The efficacy of the vaccine was evaluated in a susceptible hamster model in terms of the humoral immune response and survival from leptospiral challenge. Four weeks of oral administration of live E. coli expressing LigA7-13 improved survival from intraperitoneal (i.p.) and intradermal (i.d.) challenge by L. interrogans serovar Copenhageni strain Fiocruz L1-130 in Golden Syrian hamsters. Immunization with E. coli expressing LigA7-13 resulted in a systemic antibody response, and a significant LigA7-13 IgG level after the first 2 weeks of immunization was completely predictive of survival 28 days after challenge. As in previous LigA vaccine studies, all immunized hamsters that survived infection had renal leptospiral colonization and histopathological changes. In summary, an oral LigA-based vaccine improved survival from leptospiral challenge by either the i.p. or i.d. route.

Is therapeutic lymph node dissection of value for lymph node recurrence in melanoma?

BACKGROUND: Therapeutic lymphadenectomy (TLND) is still performed in most melanoma patients to treat nodal recurrences after initial negative lymph node biopsy (-SLNB), despite the lack of evidence for survival benefit. We sought to compare melanoma-specific survival (MSS) and distant metastasis-free survival (DMFS) of patients who underwent TLND versus no TLND using our institutional and MSTL-1 databases. METHODS: We identified 146 patients with nodal recurrence following -SLNB: 132 underwent TLND and 14 did not. DMFS and MSS were evaluated for the cohorts followed by a matched-pair analysis between the cohorts. RESULTS: No difference was observed in five-year DMFS (p â€‹= â€‹0.454) and five-year MSS (p â€‹= â€‹0.945) between the two groups. The matched-pair analysis showed similar results (p â€‹= â€‹0.329 and p â€‹= â€‹0.363 for DMSF and MSS, respectively). CONCLUSIONS: From this limited retrospective study, TLND for nodal recurrence after a -SLNB does not appear to improve DMFS or MSS in melanoma patients compared to no TLND.

Implications of tumor-positive sentinel lymph nodes in single vs multiple nodal basins in melanoma.

Background: Melanoma patients' prognosis is based on the primary tumor characteristics and the tumor status of the regional lymph nodes. The advent of lymphoscintigraphy with SLN biopsy (SLNB) has shown that melanoma can drain to multiple nodal basins but the significance of multiple basins (vs. one basin) with tumor-positive sentinel lymph node(s) (+SLN) of similar tumor burden has not been shown. We examined the impact of the number of nodal basins with +SLN (+basin) in melanoma patients and its significance for patients' prognosis and survival. Study design: We identified 1,915 patients with +SLN from two randomized surgical clinical trials: Multicenter Selective Lymphadenectomy Trials I and II. Patient groups were divided based on number of +SLNs and number of +basins. Disease-free survival (DFS), distant disease-free survival (DDFS) and melanoma-specific survival (MSS) were compared with the Kaplan-Meier method and log-rank tests. Univariable and multivariable analyses were performed using Cox proportional hazard regressions. Results: Among the 1,915 patients, 1,501 had only one +SLN (78%) in one basin and 414 (22%) had multiple +SLNs: 340 located in one basin and 74 in multiple basins. Among patients with multiple +SLNs, those with multiple +basins have a worse DFS, DDFS and MSS than those with a single basin (p ≤ 0.0001 for all comparisons). MSS was significantly different based on AJCC stages: AJCC IIIA and IIIB (p ≤ 0.001 and 0.0287, respectively). Conclusion: Our results suggest that the number of tumor-positive basins may be important for staging and in understanding the biology of lymph node metastases.

Leptospira interrogans catalase is required for resistance to H2O2 and for virulence.

Pathogenic Leptospira spp. are likely to encounter higher concentrations of reactive oxygen species induced by the host innate immune response. In this study, we characterized Leptospira interrogans catalase (KatE), the only annotated catalase found within pathogenic Leptospira species, by assessing its role in resistance to H(2)O(2)-induced oxidative stress and during infection in hamsters. Pathogenic L. interrogans bacteria had a 50-fold-higher survival rate under H(2)O(2)-induced oxidative stress than did saprophytic L. biflexa bacteria, and this was predominantly catalase dependent. We also characterized KatE, the only annotated catalase found within pathogenic Leptospira species. Catalase assays performed with recombinant KatE confirmed specific catalase activity, while protein fractionation experiments localized KatE to the bacterial periplasmic space. The insertional inactivation of katE in pathogenic Leptospira bacteria drastically diminished leptospiral viability in the presence of extracellular H(2)O(2) and reduced virulence in an acute-infection model. Combined, these results suggest that L. interrogans KatE confers in vivo resistance to reactive oxygen species induced by the host innate immune response.

Inactivation of clpB in the pathogen Leptospira interrogans reduces virulence and resistance to stress conditions.

Leptospira interrogans is the causative agent of leptospirosis, which is an emerging zoonotic disease. Resistance to stress conditions is largely uncharacterized for this bacterium. We therefore decided to analyze a clpB mutant that we obtained by random transposon mutagenesis. The mutant did not produce any of the two isoforms of ClpB. The clpB mutant exhibited growth defects at 30° and 37°C and in poor nutrient medium and showed increased susceptibility to oxidative stress, whereas the genetically complemented strain was restored in ClpB expression and in vitro wild-type growth. We also showed that the clpB mutant was attenuated in virulence in an animal model of acute leptospirosis. Our findings demonstrate that ClpB is involved in the general stress response. The chaperone is also necessary, either directly or indirectly, for the virulence of the pathogen L. interrogans.

Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria.

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-ß-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.

Transposon Sequencing in Leptospira interrogans.

Our limited understanding of the relationship of genotype to phenotype in the spirochete Leptospira interrogans stems from the inefficiency of the genetic tools available to manipulate the pathogen. The recent development of random transposon mutagenesis in L. interrogans has allowed the creation of large libraries of mutants, permitting the identification of several genes involved in certain functions such as virulence. However, the process of phenotypically screening individual mutants in the library remains time- and labor-intensive. Here, we describe a transposon sequencing technique (Tn-Seq), which combines random transposon mutagenesis with high-throughput sequencing for screening L. interrogans mutants more rapidly with fewer resources than traditional methods.

Evaluation of Vaccine Candidates against Leptospirosis using Golden Syrian Hamsters.

Leptospirosis is a major public health problem, especially in developing countries. Current vaccine studies focus on identifying Leptospira proteins that elicit protective immunity. Here, we describe a method to assess recombinant proteins for their ability to protect hamsters from fatal infection against Leptospira and to provide sterilizing immunity.

Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis.

The dynamics of leptospirosis infection have been poorly studied. The purpose of this study was to determine the LD(50), rate of bacterial dissemination, histopathology and antibody responses against leptospira following inoculation with the highly virulent Leptospira interrogans Fiocruz L1-130 strain in a guinea pig model of leptospirosis. Three routes of infection (intraperitoneal, conjunctival and subcutaneous inoculation) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in the blood and tissues of infected animals were determined over a 1 week course of infection using quantitative real-time PCR (qPCR). Bacteraemia peaked at day 5 post-infection reaching more than 5x10(4) leptospires ml(-1). The highest spirochaetal load was found in the liver and kidneys, and was associated with alterations in organ tissues and a decline in liver and kidney functions. In contrast, lesions and bacteria were not detected in guinea pigs infected with an avirulent strain derived from a high-passage-number in vitro-passaged variant of the Fiocruz L1-130 strain. The use of qPCR supports the findings of earlier studies and provides an easy and reliable method for the quantification of L. interrogans in the tissues of infected animals. qPCR will be used in future studies to evaluate the efficacy of vaccine candidates against leptospirosis and the virulence of selected L. interrogans mutants relative to the parental strain.

Immunoprotective properties of recombinant LigA and LigB in a hamster model of acute leptospirosis.

Leptospirosis is the most widespread zoonosis and is considered a major public health problem worldwide. Currently, there is no widely available vaccine against leptospirosis for use in humans. A purified, recombinant subunit vaccine that includes the last six immunoglobulin-like (Ig-like) domains of the leptospiral protein LigA (LigA7'-13) protects against lethal infection but not renal colonization after challenge by Leptospira interrogans. In this study, we examined whether the addition of the first seven Ig-like domains of LigB (LigB0-7) to LigA7'-13, can enhance immune protection and confer sterilizing immunity in the Golden Syrian hamster model of acute leptospirosis. Hamsters were subcutaneously immunized with soluble, recombinant LigA7'-13, LigB0-7, or a combination of LigA7'-13 and LigB0-7 in Freund's adjuvant. Immunization with Lig proteins generated a strong humoral immune response with high titers of IgG that recognized homologous protein, and cross-reacted with the heterologous protein as assessed by ELISA. LigA7'-13 alone, or in combination with LigB0-7, protected all hamsters from intraperitoneal challenge with a lethal dose of L. interrogans serovar Copenhageni strain Fiocruz L1-130. However, bacteria were recovered from the kidneys of all animals. Of eight animals immunized with LigB0-7, only three survived Leptospira challenge, one of which lacked renal colonization and had antibodies to native LigB by immunoblot. In addition, sera from two of the three LigB0-7 immunized survivors cross-reacted with LigA11-13, a region of LigA that is sufficient for protection. In summary, we confirmed that LigA7'-13 protects hamsters from death but not infection, and immunization with LigB0-7, either alone or in combination with LigA7'-13, did not confer sterilizing immunity.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection.

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection.

Pathogenic species of Leptospira are the causative agents of leptospirosis, a zoonotic disease that causes mortality and morbidity worldwide. The understanding of the virulence mechanisms of Leptospira spp is still at an early stage due to the limited number of genetic tools available for this microorganism. The development of random transposon mutagenesis in pathogenic strains a decade ago has contributed to the identification of several virulence factors. In this study, we used the transposon sequencing (Tn-Seq) technique, which combines transposon mutagenesis with massive parallel sequencing, to study the in vivo fitness of a pool of Leptospira interrogans mutants. We infected hamsters with a pool of 42 mutants (input pool), which included control mutants with insertions in four genes previously analyzed by virulence testing (loa22, ligB, flaA1, and lic20111) and 23 mutants with disrupted signal transduction genes. We quantified the mutants in different tissues (blood, kidney and liver) at 4 days post-challenge by high-throughput sequencing and compared the frequencies of mutants recovered from tissues to their frequencies in the input pool. Control mutants that were less fit in the Tn-Seq experiment were attenuated for virulence when tested separately in the hamster model of lethal leptospirosis. Control mutants with unaltered fitness were as virulent as the wild-type strain. We identified two mutants with the transposon inserted in the same putative adenylate/guanylate cyclase gene (lic12327) that had reduced in vivo fitness in blood, kidney and liver. Both lic12327 mutants were attenuated for virulence when tested individually in hamsters. Growth of the control mutants and lic12327 mutants in culture medium were similar to that of the wild-type strain. These results demonstrate the feasibility of screening large pools of L. interrogans transposon mutants for those with altered fitness, and potentially attenuated virulence, by transposon sequencing.

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans.

Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity. Complementation of the mutation with the sph2 gene partially restored production of hemolytic and sphingomyelinase activities. These results indicate that the sph2 gene product contributes to the hemolytic and sphingomyelinase activities secreted by L. interrogans and most likely dominates those functions under the culture condition tested.