John Kalbfleisch

Modulation of the Phosphoinositide 3-Kinase Pathway Alters Innate Resistance to Polymicrobial Sepsis

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-β, IL-2, IL-6, IL-10, IL-12, and TNF-α in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.

Serum Glucan Levels Are Not Specific for Presence of Fungal Infections in Intensive Care Unit Patients

ABSTRACT Fungal infections in the critically ill patient are difficult to diagnose and are associated with a high mortality rate. A major obstacle to managing fungal infection is the lack of a reliable clinical assay that will rapidly identify patients with fungal sepsis. Glucans are polymers of glucose that are found in the cell wall of fungi and certain bacteria. Glucans are also released from the fungal cell wall into the extracellular milieu. Several studies have reported that detection of fungal glucan in serum or plasma is useful in the diagnosis of mycoses. However, recent studies have questioned the clinical utility of this assay. In this study, we examined serum glucan levels in intensive care unit (ICU) patients and attempt to correlate serum glucan levels with the presence of fungal infection. Following attainment of informed consent, serum was harvested from 46 ICU patients with confirmed fungal infections, confirmed bacterial infections, or no evidence of infection. Sera from eight healthy volunteers served as control. Serum glucan was assayed with a glucan-specific Limulus assay. Serum glucan levels were increased (69.6 ± 17 pg/ml; P < 0.001) in ICU patients versus the normal (11.5 ± 1.3 pg/ml) and noninfected ICU (27.4 ± 17 pg/ml) controls. However, serum glucan levels were not different in patients with confirmed fungal infections versus those with confirmed bacterial infections. Thus, serum glucan levels did not show a correlation with the presence of fungal infections and do not appear to be specific for fungal infections. However, the assay may be useful as a negative predictor of infection.

Modulation of tissue Toll-like receptor 2 and 4 during the early phases of polymicrobial sepsis correlates with mortality.

OBJECTIVE To determine whether there was a correlation between induction of polymicrobial sepsis, modulation of tissue Toll-like receptor (TLR) gene, and protein expression and survival outcome. DESIGN Prospective, randomized animal study. SETTING University medical school research laboratory. SUBJECTS Age- and weight-matched ICR/HSD mice. INTERVENTIONS Sepsis was induced by cecal ligation and puncture (CLP). No-surgery and sham (laparotomy)-operated mice were controls. We also examined tissue TLR2 and TLR4 messenger RNA and TLR4 protein levels in mice treated with an immunomodulator that increases survival in polymicrobial sepsis. In the immunomodulator study, mice were treated with glucan phosphate (50 mg/kg, intraperitoneally) 1 hr before CLP. No-surgery, sham surgery, glucan + no-surgery, sham surgery + glucan, and CLP groups were employed as controls. MEASUREMENTS AND MAIN RESULTS Total RNA was isolated from liver, lung, and spleen at 0, 1, 3, 6, 8, and 24 hrs after CLP. TLR gene expression was assessed by reverse transcription-polymerase chain reaction. Tissue TLR4 protein levels were evaluated at 24 hrs by Western blot and immunohistochemistry. CLP sepsis increased (p <.05) liver and lung TLR2 and TLR4 gene expression compared with controls. TLR4 protein concentrations also were increased. Increased TLR2/4 gene and TLR4 protein expression correlated with mortality. Immunoprophylaxis with glucan phosphate increased (p <.001) long-term survival (20% vs. 70%) but inhibited (p <.05) CLP-induced increases in tissue TLR2 and TLR4 messenger RNA expression as well as TLR4 protein expression. CONCLUSIONS Early increases in TLR2/4 gene and TLR4 protein expression correlated with mortality, whereas blunting TLR gene and protein expression correlated with improved long-term survival. This suggests that early up-regulation of tissue TLR2/4 may play a role in the proinflammatory response and pathophysiology of polymicrobial sepsis.

Differential roles of TLR2 and TLR4 in acute focal cerebral ischemia/reperfusion injury in mice.

Recent studies have shown that Toll-like receptors (TLRs) are involved in cerebral ischemia/reperfusion (I/R) injury. This study was to investigate the role of TLR2 and TLR4 in acute focal cerebral I/R injury. Cerebral infarct size, neurological function and mortality were evaluated. NFsmall ka, CyrillicB binding activity, phosphorylation of Ismall ka, CyrillicBalpha, Akt and ERK1/2 were examined in ischemic cerebral tissue by EMSA and Western blots. Compared to wild type (WT) mice, in TLR4 knockout (TLR4KO) mice, brain infarct size was decreased (2.6+/-1.18% vs 11.6+/-1.97% of whole cerebral volume, p<0.05) and neurological function was maintained (7.3+/-0.79 vs 4.7+/-0.68, p<0.05). However, compared to TLR4KO mice, TLR2 knockout (TLR2KO) mice showed higher mortality (38.2% vs 13.0%, p<0.05), decreased neurological function (2.9+/-0.53 vs 7.3+/-0.79, p<0.05) and increased brain infarct size (19.1+/-1.33% vs 2.6+/-1.18%, p<0.05). NFsmall ka, CyrillicB activation and Ismall ka, CyrillicBalpha phosphorylation were attenuated in TLR4KO mice (1.09+/-0.02 and 1.2+/-0.04) compared to TLR2KO mice (1.31+/-0.02 and 2.2+/-0.32) after cerebral ischemia. Compared to TLR4KO mice, in TLR2KO mice, the phosphorylation of Akt (0.2+/-0.03 vs 0.9+/-0.16, p<0.05) and ERK1/2 (0.8+/-0.06 vs 1.3+/-0.17) evoked by cerebral I/R was attenuated. The present study demonstrates that TLR2 and TLR4 play differential roles in acute cerebral I/R injury. Specifically, TLR4 contributes to cerebral I/R injury, while TLR2 appears to be neuroprotective by enhancing the activation of protective signaling in response to cerebral I/R.

Early speech and language development in children with velocardiofacial syndrome.

Speech-language impairment is one of the most common clinical features in velocardiofacial syndrome (VCFS). This report describes the speech and language development of four children with VCFS studied longitudinally from 6 to 30 months of age and compares their performance with three groups of children: (1) normally developing children, (2) children with cleft lip and palate, and (3) children with isolated cleft palate. The data show that young children with VCFS show a receptive-expressive language impairment from the onset of language. Further, speech and expressive language development were severely delayed beyond a level predicted by their other developmental or receptive language performance. The children with VCFS showed severe limitations in speech sound inventories and early vocabulary development that far exceeded those shown by the children with cleft lip and palate and children with isolated cleft palate. This study indicates that young children with VCFS emerge from a critical speech and language learning period with severe limitations in their communicative abilities. Further studies are required to describe the later course of these early speech and language impairments and to explore the relationship to learning disabilities described for older children with VCFS. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:714-723, 1999.

Preconditioning with a TLR2 specific ligand increases resistance to cerebral ischemia/reperfusion injury

The brains resistance to ischemic injury can be transiently augmented by prior exposure to a sub-lethal stress stimulus, i.e. preconditioning. It has been reported that Toll-like receptors (TLRs) are involved in the preconditioning-induced protective effect against ischemic brain injury. In this study, we investigated the effect of preconditioning with a TLR2 specific ligand, Pam3CSK4, on focal cerebral ischemia/reperfusion (I/R) injury in mice. Pam3CSK4 was administered systemically 24 h before the mice were subjected to focal cerebral ischemia (1 h) followed by reperfusion. Cerebral infarct size was determined, blood brain barrier (BBB) permeability was evaluated, and expression of tight-junction proteins were examined after focal cerebral I/R. Results showed that pre-treatment with Pam3CSK significantly reduced brain infarct size (1.9+/-0.5% vs 9.4+/-2.2%) compared with the untreated I/R group. Pam3CSK4 pre-treatment also significantly reduced acute mortality (4.3% vs 24.2%), preserved neurological function (8.22+/-0.64 vs 3.91+/-0.57), and attenuated brain edema (84.61+/-0.08% vs 85.29+/-0.09%) after cerebral I/R. In addition, Pam3CSK4 pre-treatment preserved BBB function as evidenced by decreased leakage of serum albumin (0.528+/-0.026 vs 0.771+/-0.059) and Evans Blue (9.23+/-0.72 microg/mg vs 12.56+/-0.65 microg/mg) into brain tissue. Pam3CSK4 pre-treatment also attenuated the loss of the tight junction protein occludin in response to brain I/R injury. These results suggest that TLR2 is a new target of ischemic preconditioning in the brain and preconditioning with a TLR2 specific ligand will protect the brain from I/R injury.

Normal Human Fibroblasts Express Pattern Recognition Receptors for Fungal (1→3)-β-d-Glucans

ABSTRACT Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity. Glucans are thought to mediate their effects via interaction with membrane receptors on macrophages, neutrophils, and NK cells. There have been no reports of glucan receptors on nonimmune cells. We investigated the binding of a water-soluble glucan in primary cultures of normal human dermal fibroblasts (NHDF). Membranes from NHDF exhibited saturable binding with an apparent dissociation constant (KD) of 8.9 ± 1.9 μg of protein per ml and a maximum binding of 100 ± 8 resonance units. Competition studies demonstrated the presence of at least two glucan binding sites on NHDF. Glucan phosphate competed for all binding sites, with a KD of 5.6 μM (95% confidence interval [CI], 3.0 to 11 μM), while laminarin competed for 69% ± 6% of binding sites, with a KD of 3.7 μM (95% CI, 1.9 to 7.3 μM). Glucan (1 μg/ml) stimulated fibroblast NF-κB nuclear binding activity and interleukin 6 (IL-6) gene expression in a time-dependent manner. NF-κB was activated at 4, 8, and 12 h, while IL-6 mRNA levels were increased by 48% at 8 h. This is the first report of pattern recognition receptors for glucan on human fibroblasts and the first demonstration of glucan binding sites on cells other than leukocytes. It also provides the first evidence that glucans can directly modulate the functional activity of NHDF. These results provide new insights into the mechanisms by which the host recognizes and responds to fungal (1→3)-β-d-glucans and suggests that the response to glucans may not be confined to cells of the immune system.

Immunohistochemical detection of p53 protein as a prognostic indicator in prostate cancer.

Mutation of the p53 gene is the most common genetic alteration in human cancers. The mutant p53 protein is more stable than the wild type and can be detected by immunohistology. The objective of the current study was to evaluate the immunohistological detection of p53 protein in prostate cancer and its utility as a prognostic indicator. We used a monoclonal anti-p53 antibody and immunostained primary prostate adenocarcinomas (stages A1 to D1) from 109 patients with a mean follow-up of 3.8 years (range, 1.3 to 9.3 years). Immunoreactivity for p53 was seen in 23 cancers (21%). There were 12 instances of progression (14%) among the p53-negative cancers versus seven (30%) among the p53-positive group. Survival analysis using three univariate statistical tests showed that p53 reactivity (P < .03), Gleason score (P < .01), and stage (P < .05) had significant effects on time to progression of prostate cancer. Multivariate analyses showed that Gleason score was significant with all three tests; p53 reactivity was significant with the Wilcoxon test but only approached significance by the log rank and Cox tests. When the analyses included only patients with Gleason scores 2 to 7 (N = 94), univariate analyses showed that p53 reactivity was strongly related to progression of prostate cancer (P < .007). Stage also was significant (P < 0.04), but Gleason score was not. Multivariate analyses showed only p53 reactivity to be significant (P < .007). In conclusion, mutation of the p53 gene may be involved in prostate cancer carcinogenesis. p53 reactivity marks an aggressive subset of prostate cancer and appears to be an independent prognostic indicator that is particularly valuable among the low to intermediate grade cancers.

Early Activation of Hepatic Nfκb and Nf-il6 in Polymicrobial Sepsis Correlates With Bacteremia, Cytokine Expression, and Mortality

BACKGROUND The role of transcription factor activation in the pathophysiology of sepsis syndrome has not been established. This study investigated the relation between tissue nuclear factor kappaB (NFkappaB) and nuclear factor interleukin 6 (NF-IL6 or C/EBP) activation and bacteremia, inflammatory cytokine expression, and mortality in a murine model of cecal ligation and puncture (CLP). METHODS Transcription factor activation was assessed by the electrophoretic mobility shift assay. Cytokine mRNA levels were established by reverse transcription-polymerase chain reaction and quantified by scanning densitometry. Bacteremia was evaluated by standard aerobic and anaerobic microbiologic methods. RESULTS CLP stimulated hepatic NFkappaB activation at 2, 3, 4, 5, 6, and 8 hours compared with control and sham-operated mice. Hepatic NFkappaB activation during CLP peaked at 4 hours (1114% vs. no surgery, 609% vs. sham). Hepatic NF-IL6 activation was observed at 3, 4, and 6 hours after CLP. Hepatic and splenic levels of tumor necrosis factor-alpha and IL-6 mRNA were also elevated after CLP. Bacteremia in CLP mice consisted of Bacteroides species and to a lesser extent facultative gram-negative bacilli and group D Enterococcus. CONCLUSIONS Early activation of hepatic and splenic NFkappaB and NF-IL6 positively correlates with tissue cytokine mRNA expression and mortality in a surgical model of polymicrobial sepsis. The data suggest that transcription factor activation is an early event in the pathophysiology of sepsis.

TLR2 Ligand Induces Protection against Cerebral Ischemia/Reperfusion Injury via Activation of Phosphoinositide 3-Kinase/Akt Signaling

This study examined the effect of TLR2 activation by its specific ligand, Pam3CSK4, on cerebral ischemia/reperfusion (I/R) injury. Mice (n = 8/group) were treated with Pam3CSK4 1 h before cerebral ischemia (60 min), followed by reperfusion (24 h). Pam3CSK4 was also given to the mice (n = 8) 30 min after ischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The morphology of neurons in brain sections was examined by Nissl staining. Pam3CSK4 administration significantly reduced infarct size by 55.9% (p < 0.01) compared with untreated I/R mice. Therapeutic treatment with Pam3CSK4 also significantly reduced infarct size by 55.8%. Morphologic examination showed that there was less neuronal damage in the hippocampus of Pam3CSK4-treated mice compared with untreated cerebral I/R mice. Pam3CSK4 treatment increased the levels of Hsp27, Hsp70, and Bcl2, and decreased Bax levels and NF-κB–binding activity in the brain tissues. Administration of Pam3CSK4 significantly increased the levels of phospho-Akt/Akt and phospho-GSK-3β/GSK-3β compared with untreated I/R mice. More significantly, either TLR2 deficiency or PI3K inhibition with LY29004 abolished the protection by Pam3CSK4. These data demonstrate that activation of TLR2 by its ligand prevents focal cerebral ischemic damage through a TLR2/PI3K/Akt-dependent mechanism. Of greater significance, these data indicate that therapy with a TLR2-specific agonist during cerebral ischemia is effective in reducing injury.