Draft genome of Thermomonospora sp. CIT 1 (Thermomonosporaceae) and in silico evidence of its functional role in filter cake biomass deconstruction.

The filter cake from sugar cane processing is rich in organic matter and nutrients, which favors the proliferation of microorganisms with potential to deconstruct plant biomass. From the metagenomic data of this material, we assembled a draft genome that was phylogenetically related to Thermomonospora curvata DSM 43183, which shows the functional and ecological importance of this bacterium in the filter cake. Thermomonospora is a gram-positive bacterium that produces cellulases in compost, and it can survive temperatures of 60 ºC. We identified a complete set of biomass depolymerizing enzymes in the draft genome of Thermomonospora sp. CIT 1, such as α-amylase, catalase-peroxidases, ß-mannanase, and arabinanase, demonstrating the potential of this bacterium to deconstruct the components of starch, lignin, and hemicellulose. In addition, the draft genome of Thermomonospora sp. CIT 1 contains 18 genes that do not share identity with five other species of Thermomonospora, suggesting that this bacterium has different genetic characteristics than those present in genomes reported so far for this genus. These findings add a new dimension to the current understanding of the functional profile of this microorganism that inhabits agro-industrial waste, which may boost new gene discoveries and be of importance for application in the production of bioethanol.

Differential Proteomics for Distinguishing Ischemic Stroke from Controls: a Pilot Study of the SpecTRA Project.

A diagnostic blood test for stroke is desirable but will likely require multiple proteins rather than a single "troponin." Validating large protein panels requires large patient numbers. Mass spectrometry (MS) is a cost-effective tool for this task. We compared differences in the abundance of 147 protein markers to distinguish 20 acute cerebrovascular syndrome (ACVS) patients who presented to the Emergency Department of one urban hospital within < 24 h from onset) and from 20 control patients who were enrolled via an outpatient neurology clinic. We targeted proteins from the stroke literature plus cardiovascular markers previously studied in our lab. One hundred forty-one proteins were quantified using MS, 8 were quantified using antibody protein enrichment with MS, and 32 were measured using ELISA, with some proteins measured by multiple techniques. Thirty proteins (4 by ELISA and 26 by the MS techniques) were differentially abundant between mimic and stroke after adjusting for age in robust regression analyses (FDR < 0.20). A logistic regression model using the first two principal components of the proteins significantly improved discrimination between strokes and controls compared to a model based on age alone (p < 0.001, cross-validated AUC 0.93 vs. 0.78). Significant proteins included markers of inflammation (47%), coagulation (40%), atrial fibrillation (7%), neurovascular unit injury (3%), and other (3%). These results suggest the potential value of plasma proteins as biomarkers for ACVS diagnosis and the role of plasma-based MS in this area.

Plasma-Derived C1 Esterase Inhibitor for Acute Antibody-Mediated Rejection Following Kidney Transplantation: Results of a Randomized Double-Blind Placebo-Controlled Pilot Study.

Antibody-mediated rejection (AMR) is typically treated with plasmapheresis (PP) and intravenous immunoglobulin (standard of care; SOC); however, there is an unmet need for more effective therapy. We report a phase 2b, multicenter double-blind randomized placebo-controlled pilot study to evaluate the use of human plasma-derived C1 esterase inhibitor (C1 INH) as add-on therapy to SOC for AMR. Eighteen patients received 20 000 units of C1 INH or placebo (C1 INH n = 9, placebo n = 9) in divided doses every other day for 2 weeks. No discontinuations, graft losses, deaths, or study drug-related serious adverse events occurred. While the study's primary end point, a difference between groups in day 20 pathology or graft survival, was not achieved, the C1 INH group demonstrated a trend toward sustained improvement in renal function. Six-month biopsies performed in 14 subjects (C1 INH = 7, placebo = 7) showed no transplant glomerulopathy (TG) (PTC+cg≥1b) in the C1 INH group, whereas 3 of 7 placebo subjects had TG. Endogenous C1 INH measured before and after PP demonstrated decreased functional C1 INH serum concentration by 43.3% (p < 0.05) for both cohorts (C1 INH and placebo) associated with PP, although exogenous C1 INH-treated patients achieved supraphysiological levels throughout. This new finding suggests that C1 INH replacement may be useful in the treatment of AMR.

Metagenomic Assembly and Draft Genome Sequence of an Uncharacterized Prevotella sp. from Nelore Rumen.

Prevotella is one of the most abundant genera in bovine rumen, although no genome has yet been assembled by a metagenomics approach applied to Brazilian Nelore. We report the draft genome sequence of Prevotella sp., comprising 2,971,040 bp, obtained using the Illumina sequencing platform. This genome includes 127 contigs and presents a low 48% GC.

Quantifying renal allograft loss following early antibody-mediated rejection.

Unlike antibody-mediated rejection (AMR) with clinical features, it remains unclear whether subclinical AMR should be treated, as its effect on allograft loss is unknown. It is also uncertain if AMR's effect is homogeneous across donor (deceased/live) and (HLA/ABO) antibody types. We compared 219 patients with AMR (77 subclinical, 142 clinical) to controls matched on HLA/ABO-compatibility, donor type, prior transplant, panel reactive antibody (PRA), age and year. One and 5-year graft survival in subclinical AMR was 95.9% and 75.7%, compared to 96.8% and 88.4% in matched controls (p = 0.0097). Subclinical AMR was independently associated with a 2.15-fold increased risk of graft loss (95% CI: 1.19-3.91; p = 0.012) compared to matched controls, but not different from clinical AMR (p = 0.13). Fifty three point two percent of subclinical AMR patients were treated with plasmapheresis within 3 days of their AMR-defining biopsy. Treated subclinical AMR patients had no difference in graft loss compared to matched controls (HR 1.73; 95% CI: 0.73-4.05; p = 0.21), but untreated subclinical AMR patients did (HR 3.34; 95% CI: 1.37-8.11; p = 0.008). AMR's effect on graft loss was heterogeneous when stratified by compatible deceased donor (HR = 4.73; 95% CI: 1.57-14.26; p = 0.006), HLA-incompatible deceased donor (HR = 2.39; 95% CI: 1.10-5.19; p = 0.028), compatible live donor (no AMR patients experienced graft loss), ABO-incompatible live donor (HR = 6.13; 95% CI: 0.55-67.70; p = 0.14) and HLA-incompatible live donor (HR = 6.29; 95% CI: 3.81-10.39; p < 0.001) transplant. Subclinical AMR substantially increases graft loss, and treatment seems warranted.

Multiple hyperacute rejections in the absence of detectable complement activation in a patient with endothelial cell reactive antibody.

This case involves a 54-year-old patient with polycystic kidney disease and a history of hyperacute allograft rejections. Two previous compatible live donor transplants functioned immediately but failed within the first 12 h due to antibody-injury. This patient was referred for a third transplant due to decreased vascular access and progressive hypotension from uremic autonomic dysfunction. He was broadly sensitized to HLA; however, a live donor was identified through kidney paired donation for whom he had no donor-specific HLA antibody (HLA-DSA). This patient received one plasmapheresis (PP) and intravenous immunoglobulin (IVIg) treatment, anti-CD25, and anti-CD20 antibodies prior to transplant. The allograft functioned immediately but became anuric within 24 h. A biopsy revealed antibody-mediated injury in the absence of C4d. Daily PP/IVIg, a second dose of anti-CD20, and eculizumab were administered. A retrospective endothelial cell crossmatch (ECXM) was positive with serum drawn 3 days prior to transplant and these EC antibodies were enriched for IgG2 and IgG4, noncomplement activating subclasses. Postoperative day (POD) 3, HLA-DSA remained negative but a rescue splenectomy was performed. Cultured splenocytes produced antibodies that bound donor ECs but not lymphocytes. Bortezomib was initiated on POD5. Despite aggressive therapy, the allograft never regained function.

The role of nanostructure in the wetting behavior of mixed-monolayer-protected metal nanoparticles.

Self-assembled monolayer-protected nanoparticles are promising candidates for applications, such as sensing and drug delivery, in which the molecular ligands' interactions with the surrounding environment play a crucial role. We recently showed that, when gold nanoparticles are coated with a binary mixture of immiscible ligands, ordered ribbon-like domains of alternating composition spontaneously form and that their width is comparable with the size of a single solvent molecule. It is usually assumed that nanoparticles' solubility depends solely on the core size and on the molecular composition of the ligand shell. Here, we show that this is not always the case. We find that the ligand shell morphology affects the solubility of these nanoparticles almost as much as the molecular composition. A possible explanation is offered through a molecular dynamics analysis of the surface energy of monolayers differing only in their domain structure. We find that the surface free energy of such model systems can vary significantly as a function of ordering, even at fixed composition. This combined experimental and theoretical study provides a unique insight into wetting phenomena at the nano- and subnanometer scale.

Increased expression of unusual EP repeat-containing proteins in the midgut of the tsetse fly (Glossina) after bacterial challenge.

Proteins containing a glutamic acid-proline (EP) repeat epitope were immunologically detected in midguts from eight species of Glossina (tsetse flies). The molecular masses of the tsetse EP proteins differed among species groups. The amino acid sequence of one of these proteins, from Glossina palpalis palpalis, was determined and compared to the sequence of a homologue, the tsetse midgut EP protein of Glossina m. morsitans. The extended EP repeat domains comprised between 36% (G. m. morsitans) and 46% (G. p. palpalis) of the amino acid residues, but otherwise the two polypeptide chains shared most of their sequences and predicted functional domains. The levels of expression of tsetse EP protein in adult teneral midguts were markedly higher than in midguts from larvae. The EP protein was detected by immunoblotting in the fat body, proventriculus and midgut, the known major immune tissues of tsetse and is likely secreted as it was also detected in hemolymph. The EP protein was not produced by the bacterial symbionts of tsetse midguts as determined by genome analysis of Wigglesworthia glossinidia and immunoblot analysis of Sodalis glossinidius. Bacterial challenge of G. m. morsitans, by injection of live E. coli, induced augmented expression of the tsetse EP protein. The presence of EP proteins in a wide variety of tsetse, their constitutive expression in adult fat body and midguts and their upregulation after immunogen challenge suggest they play an important role as a component of the immune system in tsetse.

Increase in peripheral blood mononuclear cell (PBMC)- and CD56+ cell-mediated killing of endometrial stromal cells by mycobacteria; a possible role in endometriosis immunotherapy?

BACKGROUND: Immunological therapies have shown promising results in the treatment of endometriosis. Mycobacteria are one of the most common immune therapies used in other diseases. We have assessed the effects of mycobacteria in altering the ability of peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells to kill endometrial stromal cells using an in vitro model. This may have implications in the immunotherapy of endometriosis. METHODS: Primary cultures of endometrial stromal cells were grown from female patients and PBMCs were extracted from healthy female volunteers. Effector cells (PBMCs or NK cells) were exposed to varying concentrations of mycobacteria before their ability to kill cultured endometrial cells was tested using a 51Cr-release assay. RESULTS: Treatment of effector cells with the Connaught Substrain Bacillus of Calmette and Guérin (BCG) led to increased killing of target cells by PBMCs and NK cells. The optimal concentration for treatment of effector cells with Connaught BCG was approximately 0.1 multiplicities of infection (m.o.i.). There was a trend towards increased killing after treatment with Pasteur BCG. CD56+ (NK) cells treated with BCG at 0.1 m.o.i. showed increased killing of target cells compared with untreated effector cells. CONCLUSIONS: Endometrial stromal cells are susceptible to killer cells activated by mycobacteria. This in vitro work suggests a possible role for mycobacteria in the immunotherapy of endometriosis.

The major hormone-specific discontinuous epitopes on human chorionic gonadotrophin.

The hormone human chorionic gonadotrophin (hCG) shows extensive sequence homology with LH. Thus, many of the antigenic epitopes on hCG are shared with LH, leading to immunological cross-reaction between these two hormones. Anti-fertility and anti-cancer vaccines based upon hCG should ideally target only the hCG-specific epitopes. The hCG-unique linear epitopes located in the C-terminal peptide of the hCG beta-chain are well characterised. In contrast, the hCG-specific discontinuous epitopes, termed beta1, beta6 and beta7, have remained poorly defined. By generating hCG beta-chain molecules containing single amino acid substitutions we have identified R10, N13, R60 and Q89 as being important in the formation of the beta1 epitope, with R60 providing a particularly dominant residue. We also show that the amino acid residue Q89 contributes to the beta7 epitope, whilst D61 plays a role in both the beta6 and beta7 epitopes.

Anti-proliferative effect of mycobacteria, IFN-gamma and TNF-alpha on primary cultures derived from endometrial stroma: possible relevance to endometriosis?

PROBLEM: To assess the effects of mycobacteria and inflammatory cytokines on proliferation of endometrial stromal cells. An effect on endometrial stromal cell proliferation in vitro may suggest a similar effect on endometriotic cells in vivo. METHOD OF STUDY: Primary cultures of endometrial stromal cells were grown from female volunteers. Proliferation of cells was assessed by cell counting and incorporation of tritiated thymidine after exposure to mycobacteria or inflammatory cytokines. RESULTS: When assessed by cell counting, stromal cell growth was reduced following treatment with Connaught Bacillus of Calmette and Guérin (BCG) and Pasteur BCG: Mycobacterium smegmatis demonstrated a cytotoxic effect. Addition of the cytokines interferon (IFN)-gamma or tumour necrosis factor (TNF)-alpha at high concentrations led to a reduction in cell growth by 24 hr in two of three cell lines. A reduction in proliferation was also found when assessed by tritiated thymidine incorporation, which was statistically significant for Connaught BCG and M. smegmatis. CONCLUSIONS: Endometrial stromal cells are susceptible to the anti-proliferative effects of mycobacteria. The BCG and other mycobacteria are known immunomodulators in other disease conditions. Further work is required to assess whether these in vitro effects might translate into a useful therapy for endometriosis.

A recombinant E. coli vaccine to promote MHC class I-dependent antigen presentation: application to cancer immunotherapy.

We have examined the potential of recombinant Escherichia coli expressing listeriolysin O (LLO) to deliver tumour antigens to dendritic cells (DCs) for cancer immunotherapy. Using OVA as a model tumour antigen, we have shown in murine DCs that E. coli expressing cytoplasmic LLO and OVA proteins can deliver the OVA K(b)-restricted epitope SIINFEKL for MHC class I presentation. In contrast, when E. coli expressing OVA alone were used, MHC class II presentation of the OVA 323-339 I-A(b)-restricted peptide was predominant. When injected in vivo, DCs pulsed with E. coli expressing LLO and OVA induced production of cytotoxic T-lymphocytes capable of lysing an OVA-expressing melanoma cell line (B16-OVA) and resulted in suppression of tumour growth following challenge with B16-OVA. Immunisation of mice by direct injection of E. coli LLO/OVA provided a more potent anti-tumour response, resulting in complete protection in 75% of mice. Injection of live bacteria was not necessary as immunisation with paraformaldehyde-fixed E. coli LLO/OVA provided an even stronger anti-tumour response against B16-OVA. Altogether, our data highlight the potential of this system as a novel and efficient strategy for tumour immunotherapy.

Human dendritic cells genetically engineered to express a melanoma polyepitope DNA vaccine induce multiple cytotoxic T-cell responses.

PURPOSE: To assess the therapeutic potential of a melanoma polyepitope vaccine in human cells. Polyepitope DNA vaccines encoding T-cell epitopes have been demonstrated in murine systems to generate multiple cytotoxic T-cell responses to different antigens. Here, for the first time we demonstrate the ability of a melanoma polyepitope to stimulate lymphocytes from normal human donors to simultaneously generate multiple antigen-specific responses. EXPERIMENTAL DESIGN: Human dendritic cells (DC), transduced with a melanoma-polyepitope cDNA, were used to activate autologous lymphocytes from naïve donors as an in vitro model of DNA vaccination. Lymphocytes were primed with polyepitope or mock-transduced DC, boosted with peptide, then measured for antigen-specific cytotoxicity. RESULTS: Lymphocytes primed with polyepitope-transduced DC and boosted with peptide generated multiple cytotoxic responses. By contrast lymphocytes primed with mock-transfected DCs and boosted with peptide gave no specific cytotoxicity. However, when lymphocytes were repeatedly stimulated with polyepitope-transduced DCs immunodominance was seen with CTLs being generated to only one epitope, MART(27-35). CONCLUSIONS: We show in a human system that a melanoma polyepitope primes CTL to multiple epitopes. However, repeated stimulation by the polyepitope restricts the response to only the MART1 epitope. Thus, although polyepitope vaccines are an effective way of priming multiple naïve T-cell responses, continual boosting with polyepitope vaccines may, as a result of immunodominance, restrict the CTL. These findings have important implications for the use of DNA polyepitope vaccines in cancer immunotherapy.

The response of human dendritic cells to recombinant adenovirus, recombinant Mycobacterium bovis Bacillus Calmette Guerin and biolistic methods of antigen delivery: different induction of contact-dependant and soluble signals.

Dendritic cells (DCs) are the most potent antigen presenting cells for inducing T-cell immune responses. The ability to grow human DCs from monocyte precursors provides an abundant source of these cells, which can be modified in vitro to present antigens. Re-administration of modified DCs to patients as vaccines has been shown in some cases to induce immune responses against cancer and infectious disease. Gene delivery to DCs provides an intracellular source of antigen for efficient and persistent loading of major histocompatibility complex (MHC) class I molecules. The aim of this study was to use monocyte-derived DCs (MD-DCs) from healthy donors to compare in vitro gene transfer, mediated by adenovirus, M. bovis Bacillus Calmette Guerin (BCG) and biolistic delivery. Efficiency of transfection and effect on DC phenotype, allostimulatory capacity and cytokine secretion was investigated. Adenovirus and BCG both showed a comparable ability to transfect MD-DCs, whereas the biolistic delivery by gene gun was unsuccessful in the reporter gene delivery. BCG transfection promoted MD-DC maturation as is apparent in the surface phenotype, allostimulatory capacity and cytokine secretion from cells. In comparison, adenovirus and biolistic delivery had a reduced effect on MD-DCs although enhancement of co-stimulatory and MHC molecule expression occurred in the cells of some donors. Both BCG and adenovirus represent useful vectors for gene transfer to human DCs. The effect of BCG on DC maturation may provide additional signals for the induction of antigen-specific T-cell responses.

Expression of Interleukin-18, a Th1 cytokine, in human gastric mucosa is increased in Helicobacter pylori infection.

Interleukin-18 (IL-18), a cytokine that promotes Th1 responses, is processed to the active mature protein by caspase-1. The effects of Helicobacter pylori infection on gastric IL-18 and caspase-1 were examined. In antral mucosa, IL-18 mRNA expression was greater (P<.01) in H. pylori-positive (n=40) than in H. pylori-negative patients (n=29) with normal mucosa. Inactive precursor (24 kDa) and mature (18 kDa) IL-18 were present in antral biopsy specimens from uninfected and infected subjects. In corpus mucosa, mature IL-18 and a 16-kDa protein, corresponding to inactive IL-18, were present. Active caspase-1 p20 subunit was detected in antral and corpus mucosa of infected and uninfected subjects. These data show that, although H. pylori infection is associated with increased antral IL-18 mRNA expression, mature IL-18 protein and active caspase-1 p20 are present in mucosa of both H. pylori-infected and -uninfected subjects. IL-18 may have an important role in promoting gastric Th1 responses in H. pylori infection.

Mechanisms of action of intravesical bacille Calmette-Guérin: local immune mechanisms.

The local immune response to mycobacteria is complex, but mycobacterial antigen presentation by phagocytes to T helper cells is the pivotal interaction. Bacille Calmette-Guérin (BCG) vaccination is associated with the development of antituberculosis immunity but not necessarily with antitumor immunity. Animal studies have shown that an intact host immune system is required for the antitumor activity of BCG. Immunosuppressed and, particularly, T cell-depleted individuals fail to respond to BCG immunotherapy. Clinical and laboratory evidence suggest that the antitumor activity is concentrated at the site of BCG administration, which reinforces the view that local immune mechanisms are responsible for this phenomenon.

Diagnosis of malignant melanoma by general practitioners and hospital specialists.

The aim of this study was to audit all malignant melanomas confirmed histologically in the Scarborough Health District over six years, prompted by the continuing rise in incidence rate nationally and relatively high number of malignant melanomas excised by general practitioners (GPs) in this area. A total of 157 malignant melanomas were diagnosed (60% from females and 40% from males) over the six years; primary excisions being carried out by GPs (37%) and hospital specialists (63%). The clinical diagnosis of malignant melanoma was made in 9% of GP cases and 35% of the hospital specialist cases. However another 45.5% of GP cases, and 38% of hospital specialist cases were regarded as suspicious pigmented lesions clinically. The histological diagnosis was of superficial spreading malignant melanoma in 72% of the GP and 69% of the hospital specialist cases. Most of the GP melanomas were excised with a lateral margin of 2 mm or less (71%); around half of the hospital excisions had a margin of over 2 mm (49%). Most melanomas were 2 mm or less in depth (Breslow depth) in both the GP (81%) and hospital specialist (75%) series. Over the six year period (1993-98) the incidence of malignant melanomas has continued to rise, but Breslow depth at diagnosis has not changed significantly. It is therefore important to continue with early recognition of this condition by GPs in the first instance, reduction in its incidence being the long term goal. During five years of the study there were only 67 lesions thought clinically to be malignant melanoma (26 GP and 41 hospital specialist cases), but which proved to be benign histologically.

Role for CD40-CD40 ligand interactions in the immune response to solid tumours.

CD40-mediated interactions play an important role in the response to infections, transplantation, and cancer by affecting the development, activation, proliferation and differentiation of a variety of immune cells. In the current study we examined the role of CD40-mediated interactions in immune responses to bladder, pancreatic and breast carcinomas as well as melanoma cell lines using soluble human CD40L (rhCD40L) or anti-CD40 mAb in vitro. CD40 expression was readily detected in a large proportion of the cell lines and was augmented but not induced de novo by treatment with IFNgamma. Treatment of CD40-positive cell lines with rhCD40L or anti-CD40mAb enhanced cell surface expression of ICAM-1 and FAS and stimulated the production of IL-6, IL-8, GROalpha, GM-CSF and TNFalpha but not IL-4, IL-10, TGFbeta, MCP-1, RANTES, MIP-1beta, or IP-10. In addition, incubation of CD40+ tumour cell lines with immobilised rhCD40L or anti-CD40 mAb in vitro resulted in significant inhibition of proliferation and a corresponding decrease in viability. This CD40-mediated inhibition of cell growth was due, at least in part, to alterations in cell cycle and the induction of apoptosis. Transfection of CD40-negative tumour cell lines with the cDNA for CD40 conferred responsiveness to rhCD40L and anti-CD40 antibody. Finally, the presence of CD40 on the surface of carcinoma lines was found to be an important factor in the generation of tumour-specific T cell responses.