Basel K. al-Ramadi

Prospective evaluation of mannan and anti-mannan antibodies for diagnosis of invasive Candida infections in patients with neutropenic fever.

The diagnostic performance and usefulness of the Platelia antigen and antibody test (Bio-Rad) was investigated in a prospective study of haematological patients at risk for invasive Candida infections. Among 100 patients, 86 were eligible, of whom invasive candidiasis (IC) occurred in 12 (14%), according to the criteria of the European Organization for Research and Treatment of Cancer/Mycoses Study Group. These included candidaemia due to Candida albicans (one patient) or Candida tropicalis (four patients), and hepatosplenic candidiasis (seven patients). The comparator group of 74 patients included 50 with febrile neutropenia alone and 24 with mould infections. A strategy was developed to determine diagnostic cut-offs from receiver operating characteristic curves with maximal sensitivity and, given this sensitivity, maximal specificity, both being greater than 0. In this patient population, these values were 0.25 ng ml(-1) for mannan (M) and 2.6 arbitrary units ml(-1) for anti-mannan (AM), which are lower than those recommended by the manufacturer. All patients developed at least one positive diagnostic M or AM result during the 10 days of persistent febrile neutropenia (PFN). The optimal overall performance was found when two consecutive positive tests for both M and AM were used [sensitivity, specificity, positive predictive value and negative predictive value (NPV) (95 % confidence intervals) of 0.73 (0.39-0.94), 0.80 (0.69-0.89), 0.36 (0.17-0.59) and 0.95 (0.86-0.99), respectively]. There was a positive correlation of M with beta-D-glucan (r=0.28, P=0.01). The first positive M test was found up to a mean+/-sd of 8.8+/-8.5 (range 2-23) days prior to a clinical/mycological diagnosis of IC. Day-to-day variation in quantitative M levels was high. High-level AM responses were delayed until leucopenia resolved. The low specificities of the test performance may have been due to some of the comparator patients having subclinical Candida infections as evidenced by the high incidence of colonization among them (60% had a colonization index of >or=0.5). The high NPVs suggest that the tests may be particularly useful in excluding IC. It is feasible to explore the use of serial measurements of M and AM as part of a broader diagnostic strategy for selecting PFN patients to receive antifungal drug therapy.

Potent anti-tumor activity of systemically-administered IL2-expressing Salmonella correlates with decreased angiogenesis and enhanced tumor apoptosis.

Salmonella enterica serovar Typhimurium (hereafter S. typhimurium) stains have been shown to exert a potent inhibitory effect on the growth of human and mouse tumors in experimental models. Our laboratory has previously demonstrated that an attenuated strain of S. typhimurium engineered to express IL2 (designated strain GIDIL2) has demonstrable immunopotentiating properties, particularly affecting the innate arm of the immune system. In the present study, we wished to explore the properties of IL2-expressing Salmonella as an oncolytic agent in the highly tumorigenic B16F1 melanoma mouse model and shed light on its mechanism of action. Our data demonstrate that the systemic administration of a single dose of GIDIL2, two weeks post B16F1 implantation, had a significantly superior effect than its parental, non cytokine-expressing, strain (known as BRD509E). The improved response, which was dependent on the bacterial dose used, was observed in terms of stronger inhibition of tumor growth as well as enhanced host survival. The GIDIL2-induced anti-tumor response was correlated with decreased angiogenesis and increased necrosis within the tumor tissue. A treatment regimen involving multiple low doses of GIDIL2 was more efficacious than a single high dose regimen, resulting in extension of animal survival well beyond the normal 30 day post implantation period typically observed in this aggressive melanoma tumor model. This supports the notion of using cytokine-expressing attenuated Salmonella organisms in cancer therapy.

Recombinant human interleukin 11 and bacterial infection in patients with haemological malignant disease undergoing chemotherapy: a double-blind placebo-controlled randomised trial

Summary Background Bacteraemia in patients with haemological malignant disease causes substantial morbidity. Recombinant human interleukin 11 (rhIL-11) prevents gastrointestinal epithelial disintegrity and has immunomodulatory actions. Our aim was to ascertain whether or not treatment with rhIL-11 can prevent gut-associated infections. Methods We did a double-blind placebo-controlled randomised trial, to which we enrolled 40 patients with haemological malignant disease who were undergoing chemotherapy. Patients received either rhIL-11 50 g/kg (n=20) or placebo (n=20) daily by subcutaneous injection from the day before the start of chemotherapy until resolution of neutropenia or for 21 days, whichever was longer. Our primary outcome measure was a reduction in bacteraemia. Analysis was by intention to treat. Findings Significantly fewer patients who received rhIL-11 rather than placebo developed bacteraemia, particularly of gastrointestinal origin: the proportion of patients with at least one positive blood culture was 0·65 and 0·25, respectively (p=0·02). The numbers of patients (placebo vs rhIL-11) for each number of distinct isolates were: no organism isolated seven versus 15, one organism nine versus four, two organisms two versus one, three organisms one versus none, and four organisms one versus none (p=0·01), suggesting a lower bacterial load in the rhIL-11 than in the placebo group. Time to first bacteraemic event was longer in patients who received rhIL-11 (p=0·03) than in those who received placebo Interpretation rhIL-11 reduces the frequency and load of bacteraemia in patients with haemological malignant disease undergoing chemotherapy, possibly by gastrointestinal cytoprotective or immunological mechanisms.

Intravenous Administration of Manuka Honey Inhibits Tumor Growth and Improves Host Survival When Used in Combination with Chemotherapy in a Melanoma Mouse Model

Manuka honey has been recognized for its anti-bacterial and wound-healing activity but its potential antitumor effect is poorly studied despite the fact that it contains many antioxidant compounds. In this study, we investigated the antiproliferative activity of manuka honey on three different cancer cell lines, murine melanoma (B16.F1) and colorectal carcinoma (CT26) as well as human breast cancer (MCF-7) cells in vitro. The data demonstrate that manuka honey has potent anti-proliferative effect on all three cancer cell lines in a time- and dose-dependent manner, being effective at concentrations as low as 0.6% (w/v). This effect is mediated via the activation of a caspase 9-dependent apoptotic pathway, leading to the induction of caspase 3, reduced Bcl-2 expression, DNA fragmentation and cell death. Combination treatment of cancer cells with manuka and paclitaxel in vitro, however, revealed no evidence of a synergistic action on cancer cell proliferation. Furthermore, we utilized an in vivo syngeneic mouse melanoma model to assess the potential effect of intravenously-administered manuka honey, alone or in combination with paclitaxel, on the growth of established tumors. Our findings indicate that systemic administration of manuka honey was not associated with any alterations in haematological or clinical chemistry values in serum of treated mice, demonstrating its safety profile. Treatment with manuka honey alone resulted in about 33% inhibition of tumor growth, which correlated with histologically observable increase in tumor apoptosis. Although better control of tumor growth was observed in animals treated with paclitaxel alone or in combination with manuka honey (61% inhibition), a dramatic improvement in host survival was seen in the co-treatment group. This highlights a potentially novel role for manuka honey in alleviating chemotherapy-induced toxicity.

Influence of Vector-Encoded Cytokines on Anti-Salmonella Immunity: Divergent Effects of Interleukin-2 and Tumor Necrosis Factor Alpha

ABSTRACT Attenuated Salmonella strains are of interest as new vaccine candidates and as vectors of cloned genes of other organisms. Attenuated strains expressing specific cytokines were constructed as a means of manipulating the immune response in various disease settings. In the present study, interleukin-2 (IL-2)-expressing (GIDIL2) or tumor necrosis factor alpha (TNF-α)-expressing (GIDTNF) strains were compared with the parent strain (BRD509) for the effect of cytokines on anti-Salmonella immunity. Expression of IL-2 resulted in a rapid clearance of the organism soon after vaccination. The reduction in GIDIL2 CFU was 50- to 300-fold higher than that of BRD509 and correlated with a markedly decreased splenomegaly. Furthermore, no evidence for any significant activation, including upregulation of surface markers and production of nitric oxide (NO), was observed in spleens of GIDIL2-injected mice. In contrast, the host response to GIDTNF was marked by an early, strong, splenic cellular influx, but surprisingly, the degree of induced splenomegaly and NO secretion was only 50% of that observed in BRD509-treated mice. Despite this, bacterial colonization of the spleen in GIDTNF-immunized animals was either slightly decreased from or equivalent to that of the BRD509-treated group, suggesting the induction of additional antimicrobial mechanisms by TNF-α. In vivo protection studies demonstrated that, at limiting doses, GIDIL2 was inferior to GIDTNF and BRD509 in its capacity to protect against virulent challenge. At high doses, however, all three strains exhibited equal protective efficacy. These results demonstrate that the immune response against intracellular bacteria can be manipulated by pathogen-expressed cytokines and open the way for further fine tuning of immune responses not only to Salmonella strains themselves but also to the heterologous gene(s) carried by them.

CD154 Is Essential for Protective Immunity in Experimental Salmonella Infection: Evidence for a Dual Role in Innate and Adaptive Immune Responses

CD40-CD154 interactions are of central importance in the induction of humoral and cellular immune responses. In the present study, CD154-deficient (CD154−/−) mice were used to assess the role of CD40-CD154 interactions in regulating the immune response to a systemic Salmonella infection. Compared with C57BL/6 (CD154+/+) controls, CD154−/− mice were hypersusceptible to infection by an attenuated strain of Salmonella enterica serovar Typhimurium (S. typhimurium), as evidenced by decreased survival rate and mean time to death, which correlated with increased bacterial burden and persistence in target organs. CD154−/− mice exhibited a defect both in the production of IL-12, IFN-γ, and NO during the acute phase of the disease and in the generation of Salmonella-specific Ab responses and Ig isotype switching. Furthermore, when CD154−/− animals were administered a sublethal dose of attenuated S. typhimurium and subsequently challenged with a virulent homologous strain, all mice succumbed to an overwhelming infection. Similar treatment of CD154+/+ mice consistently resulted in ≥90% protection. The lack of protective immunity in CD154−/− mice correlated with a decreased T cell recall response to Salmonella Ags. Significant protection against virulent challenge was conferred to presensitized CD154−/− mice by transfer of serum or T cells from immunized CD154+/+ mice. For best protection, however, a combination of immune serum and T cells was required. We conclude that intercellular communications via the CD40-CD154 pathway play a critical role in the induction of type 1 cytokine responses, memory T cell generation, Ab formation, and protection against primary as well as secondary Salmonella infections.

A missense mutation in TFRC, encoding transferrin receptor 1, causes combined immunodeficiency

Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. TfrcY20H/Y20H mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.

Induction of innate immunity by IL-2-expressing Salmonella confers protection against lethal infection

Previously, we demonstrated that an attenuated Salmonella strain expressing IL-2 (designated GIDIL2) is cleared from the circulation at a much faster rate than the non-cytokine-expressing parental strain (designated BRD509). These findings suggested that IL-2 expression led to the rapid induction of innate immune responses that, in turn, accounted for the accelerated rate of bacterial clearance. In the present study, the mechanism by which this early antibacterial response is mediated was investigated. We demonstrate that as early as 2 h after infection with GIDIL2, but not BRD509, peritoneal excudate cells exhibited enhanced NK cytotoxic activity and upregulated NOS2 mRNA and NO production. This early response coincided with an enhancement of GIDIL2 clearance from the peritoneal cavity, first evidenced 22 h post-infection. Moreover, it conferred a high level of protection against virulent Salmonella challenge given within 16-20 h of GIDIL2 administration. These findings highlight the importance of innate immunity in the control of early bacterial proliferation and demonstrate the rapidity by which these responses are induced following bacterial entry.

Cytokine expression by attenuated intracellular bacteria regulates the immune response to infection: the Salmonella model

Attenuated Salmonella strains have shown excellent efficacy as mucosal vaccine delivery systems. In the present report, several recombinant strains of Salmonella enterica serovar Typhimurium, engineered to express defined murine cytokines, were used to study their potential immunoregulatory capacity in the mouse model of typhoid fever. Specifically, recombinant strains expressing IL-2 (known as GIDIL2) or TNF-alpha (GIDTNF) were compared with the parental, non-cytokine-secreting, strain (BRD509) for their ability to induce a variety of immune responses in susceptible BALB/c mice. Our findings indicate that bacterially-expressed cytokines are functional in vivo and do induce a unique pattern of responses, quite distinct from that induced by BRD509 organisms. Both the type and magnitude of specific immune parameters were affected. These included the capacity to induce an inflammatory response resulting in a state of profound splenomegaly and hepatomegaly, activation of individual immune cells (particularly macrophages and other myeloid lineage cells), and the induction of nitric oxide (NO) secretion. Furthermore, a structural analysis using light as well as electron microscopy was undertaken to examine the host cellular response to infection with the different bacterial strains. The results indicate that cytokine expression by the invading pathogen can dramatically influence host immunity from a very early stage following infection. These findings may well have important consequences for the potential utilization of bacterial vector-encoded cytokines in immunoregulation in different disease settings.

The Src-Protein Tyrosine Kinase Lck Is Required for IL-1-Mediated Costimulatory Signaling in Th2 Cells

Src-protein tyrosine kinases are intimately involved in TCR-initiated signaling in T lymphocytes. One member of this family, Lck, is also involved in CD28-mediated costimulation in Th1 cells. In Th2 lymphocytes, the costimulatory signal can also be provided by the interaction of IL-1 with type I IL-1R (IL-1RI), culminating in the activation of NF-κB transcription factors. Proximal steps in the IL-1R pathway, however, remain poorly understood, and there is conflicting evidence as to the importance of tyrosine phosphorylation in IL-1R signaling. We have addressed this issue by examining the ability of IL-1 to costimulate the activation of Lck-deficient Th2 cells. Our data demonstrate that, in the absence of Lck, the IL-1 costimulatory pathway is blocked despite the expression of normal levels of IL-1RI. Moreover, the block is associated with a defective degradation of IκB-α and an incomplete activation of NF-κB heterodimeric complexes. Protein expression of NF-κB monomers, including p50, p65, and c-Rel, is equivalent in both wild-type and Lck-deficient Th2 cell clones. Finally, we demonstrate that, in normal Th2 cells, stimulation with IL-1 leads to a rapid induction in tyrosine phosphorylation of several substrates including Lck itself. These findings strongly suggest that Lck is required for signaling in the IL-1 costimulatory pathway in Th2 lymphocytes.