Glycyrrhizin Interacts with TLR4 and TLR9 to Resolve P. aeruginosa Keratitis.

This study tests the mechanism(s) of glycyrrhizin (GLY) protection against P. aeruginosa keratitis. Female C57BL/6 (B6), TLR4 knockout (TLR4KO), myeloid specific TLR4KO (mTLR4KO), their wildtype (WT) littermates, and TLR9 knockout (TLR9KO) mice were infected with P. aeruginosa KEI 1025 and treated with GLY or PBS onto the cornea after infection. Clinical scores, photography with a slit lamp, RT-PCR and ELISA were used. GLY effects on macrophages (Mϕ) and polymorphonuclear neutrophils (PMN) isolated from WT and mTLR4KO and challenged with KEI 1025 were also tested. Comparing B6 and TLR4KO, GLY treatment reduced clinical scores and improved disease outcome after infection and decreased mRNA expression levels in cornea for TLR4, HMGB1, and RAGE in B6 mice. TLR9 mRNA expression was significantly reduced by GLY in both mouse strains after infection. GLY also significantly reduced HMGB1 (B6 only) and TLR9 protein (both B6 and TLR4KO). In TLR9KO mice, GLY did not significantly reduce clinical scores and only slightly improved disease outcome after infection. In these mice, GLY significantly reduced TLR4, but not HMGB1 or RAGE mRNA expression levels after infection. In contrast, in the mTLR4KO and their WT littermates, GLY significantly reduced corneal disease, TLR4, TLR9, HMGB1, and RAGE corneal mRNA expression after infection. GLY also significantly reduced TLR9 and HMGB1 corneal protein levels in both WT and mTLR4KO mice. In vitro, GLY significantly lowered mRNA expression levels for TLR9 in both Mϕ and PMN isolated from mTLR4KO or WT mice after incubation with KEI 1025. In conclusion, we provide evidence to show that GLY mediates its effects by blocking TLR4 and TLR9 signaling pathways and both are required to protect against disease.

The functional significance behind expressing two IL-8 receptor types on PMN.

PMN are critical to innate immunity and are fundamental to antibacterial defense. To localize to sites of infection, PMN possess receptors that detect chemoattractant stimuli elicited at the site, such as chemokines, complement split products, or bioactive lipids. Signaling through these receptors stimulates chemotaxis toward the site of infection but also activates a number of biochemical processes, with the result that PMN kill invading bacteria. PMN possess two receptors, CXCR1 and CXCR2, for the N-terminal ELR motif-containing CXC chemokines, although only two chemokine members bind both receptors and the remainder binding only CXCR2. This peculiar pattern in receptor specificity has drawn considerable interest and investigation into whether signaling through each receptor might impart unique properties on the PMN. Indeed, at first glance, CXCR1 and CXCR2 appear to be functionally redundant; however, there are differences. Considering these proinflammatory activities of activating PMN through chemokine receptors, there has been great interest in the possibility that blocking CXCR1 and CXCR2 on PMN will provide a therapeutic benefit. The literature examining CXCR1 and CXCR2 in PMN function during human and modeled diseases will be reviewed, asking whether the functional differences can be perceived based on alterations in the role PMN play in these processes.

Therapeutic effect of blocking CXCR2 on neutrophil recruitment and dextran sodium sulfate-induced colitis.

Dextran sodium sulfate (DSS)-induced colitis in mice is characterized by polymorphonuclear neutrophil (PMN) infiltration into the colonic mucosa and lumen. The mechanism by which this occurs is unclear. To begin to understand the mechanism, we determined the role of the PMN chemokine receptor, CXCR2, in DSS-induced colitis by using CXCR2(-/-) mice or by neutralizing CXCR2. DSS was administered through drinking water to CXCR2(-/-) and BALB/c mice for 5 days followed by regular water for 1 day. In the neutralization study, mice were injected with control serum or goat anti-CXCR2 antiserum. BALB/c mice receiving DSS and control serum-injected mice receiving DSS lost weight and showed considerable clinical illness. Histological observation revealed submucosal edema, PMN infiltration into the submucosa and mucosa, extensive crypt damage with abscesses, and ulceration. In contrast, both the CXCR2(-/-) and anti-CXCR2 antiserum-treated mice gained weight and had significantly lower symptom scores. Histology of these mice showed submucosal edema but relatively intact crypt architecture and very few ulcers. Significantly fewer PMNs were found in the mucosa in anti-CXCR2 anti-serum compared with control serum-injected inflamed mice, but no significant difference in eosinophil infiltration was observed between the groups. Our experiments identify a role for CXCR2 in DSS-induced colitis and suggest that antagonizing CXCR2 provides some therapeutic efficacy, possibly by impeding PMN recruitment into the mucosa. Antagonizing CXCR2 may form the basis for therapeutic drugs directed at controlling colitis.

Epidemiological studies on pulmonary pathogens in HIV-positive and -negative subjects with or without community-acquired pneumonia with special emphasis on Mycoplasma pneumoniae.

The prevalence of Mycoplasma pneumoniae among HIV-positive patients with community-acquired pneumonia (CAP) remains unclear. We investigated 300 HIV-positive adults (200 with CAP and 100 with no respiratory illness) and 75 HIV-negative adults with CAP for the prevalence of respiratory pathogens using culture and serology. A growth inhibition test was employed to confirm the isolates of M. pneumoniae using species-specific typing sera. The prevalence of M. pneumoniae in HIV-positive subjects was 17% by induced sputum and 11.3% by throat swab culture. The seroprevalence of anti-M. pneumoniae IgM was 11.7% by ELISA and 14.3% by the gelatin microparticle agglutination test. The prevalence of M. pneumoniae among HIV-negative cases was relatively low. Streptococcus pneumoniae was predominant (28%) among subjects with lower respiratory disease, whereas Staphylococcus aureus (15%) was common among upper respiratory symptomatic cases. Rales (P=0.001), pharyngeal erythema (P=0.02), cervical adenopathy (P=0.004), skin rash (P=0.001), and crepitations (P=0.001) were each significantly related to M. pneumoniae positivity. Statistical significance was observed in relation to total lymphocyte count (P=0.02) and erythrocyte sedimentation rate (P=0.04), as well as M. pneumoniae positivity. This study shows that the prevalence of M. pneumoniae in HIV-positive subjects is comparatively higher than in HIV-negative subjects with pulmonary symptoms, and concords with previous pilot studies carried out in Chennai, South India.

Investigation on the early events of apoptosis in senescent erythrocytes with special emphasis on intracellular free calcium and loss of phospholipid asymmetry in chronic renal failure.

BACKGROUND: The pathophysiological link between increased blood concentrations of factors responsible for the derangement and erythrocyte membrane functions in chronic renal failure (CRF) patients are not thoroughly elucidated. We studied the erythrocyte characteristics and phospholipid asymmetry loss in CRF patients with different grades of uremia and also examined the involvement of intracellular free Ca(2+) in early events of apoptosis in uremic erythrocytes. METHODS: The studied population consisted of 90, age and sex matched control subjects (Group I) and 238 CRF cases divided into 3 groups (Group II, III and IV) according to urea concentrations and complexity of secondary complications. Erythrocyte membrane fluidity determined by binding of MC540. Intracellular free Ca(2+) concentration was determined by the 2-wavelength method by using fluorescent calcium-sensitive probe FURA-2AM. Measurement of erythrocyte phosphatidylserine exposure by flow cytometry using Annexin V-FITC. RESULTS: Cholesterol shedding increased with increasing severity of uremic complications. Erythrocytes from Group II show mild echinocyte or formation of spicules on the erythrocyte membrane surface whereas in Group III and IV they were echinocytic. Binding of MC540 was significantly higher with progression of uremic complications. Surface charge of uremic erythrocyte membrane was significantly reduced when compared with control subjects. Intracellular free Ca(2+) was positively correlated with binding of MC540 and surface hydrophobicity. The phosphatidylserine exposure of erythrocytes was significantly higher (p<0.001) in uremic patients when compared with controls. CONCLUSIONS: Phosphatidylserine (PS) exposure erythrocytes were significantly increased in uremic patients when compared with controls. Uremic complications predisposes to membrane damages in erythrocytes.

Oxalate mediated nephronal impairment and its inhibition by c-phycocyanin: a study on urolithic rats.

The assumption of oxidative stress as a mechanism in oxalate induced renal damage suggests that antioxidants might play a beneficial role against oxalate toxicity. An in vivo model was used to investigate the effect of C-phycocyanin (from aquatic micro algae; Spirulina spp.), a known antioxidant, against calcium oxalate urolithiasis. Hyperoxaluria was induced in two of the 4 groups of Wistar albino rats (n = 6 in each) by intraperitoneally injecting sodium oxalate (70 mg/kg body weight). A pretreatment of phycocyanin (100 mg/kg body weight) as a single oral dosage was given, one hour prior to oxalate challenge. An untreated control and drug control (phycocyanin alone) were employed. Phycocyanin administration resulted in a significant improvement (p < 0.001) in the thiol content of renal tissue and RBC lysate via increasing glutathione and reducing malondialdehyde levels in the plasma of oxalate induced rats (p < 0.001), indicating phycocyanin's antioxidant effect on oxalate mediated oxidative stress. Administering phycocyanin after oxalate treatment significantly increased catalase and glucose-6-phosphate dehydrogenase activity (p < 0.001) in RBC lysate suggesting phycocyanin as a free radical quencher. Assessing calcium oxalate crystal retention in renal tissue using polarization microscopy and renal ultrastructure by electron microscopy reveals normal features in phycocyanin-- pretreated groups. Thus the study presents positive pharmacological implications of phycocyanin against oxalate mediated nephronal impairment and warrants further work to tap this potential aquatic resource for its medicinal application.

Credentials of Spirulina diet on stability and flux related properties on the biomineralization process during oxalate mediated renal calcification in rats.

BACKGROUND: High Spirulina diet is a potential risk factor for nephrolithiasis since it has the capacity to increase urinary oxalate and uric acid level, facilitating lithogenesis. Our aim was to identify the effect of Spirulina diet during hyperoxaluric condition in Wistar albino rats. METHODS: The animals were divided into four groups: control (Gl, n=6); ethylene glycol (EG) induced (G2, n=6); EG+Spirulina (G3, n=6); Spirulina alone (G4, n=6). EG at 0.75% was administered to G2 and G3 through drinking water for 4 weeks and Spirulina 1500 mg/kg feed was administered to G3 and G4. RESULTS: Urinary parameters like oxalate, uric acid, calcium, urea, and creatinine (P<0.001) were found increased after Spirulina diet under hyperoxaluric conditions compared to the same without Spirulina diet. Similarly the BUN, plasma contents of uric acid, urea, creatinine (P<0.001) were found to be raised in G3. The renal and RBC GSH levels, as estimated by HPLC, seemed decreased when compared to G2. CONCLUSIONS: The present study shows that free radicals aid in the progression of nephrolithiasis. The crystal deposition was found to be high in the renal cells of G3 than G2 and TEM revealed damage in renal cell of G3 implying that the disease deteriorates by free radical injury. In contrast the Spirulina diet alone (G4) did not induce any features relating to stone forming condition suggesting that free radical release might have been suppressed due to enrichment of dietary antioxidants and vitamins. Thus the present investigation demonstrates that during hyperoxaluric conditions the Spirulina diet must possibly be avoided and can be considered in normal subjects checked for family history of renal stone deposition.

Salubrious effect of C-phycocyanin against oxalate-mediated renal cell injury.

BACKGROUND: C-phycocyanin, a biliprotein pigment found in some blue green algae (Spirulina platensis) with nutritional and medicinal properties, was investigated for its efficacy on sodium oxalate-induced nephrotoxicity in experimentally induced urolithic rats. METHODS: Male Wistar rats were divided into four groups. Hyperoxaluria was induced in two of these groups by intraperitoneal infusion of sodium oxalate (70 mg/kg), and a pretreatment of phycocyanin (100 mg/kg) as a single oral dosage was given to one of these groups by 1 h prior to sodium oxalate infusion challenges. The study also encompasses an untreated control group and a phycocyanin-alone treated drug control group. The extent of lipid peroxidation (LPO) was evaluated in terms of renal concentrations of MDA, conjugated diene and hydroperoxides. The following assay was performed in the renal tissue (a) antioxidant enzymes such as superoxide dismutase (SOD) and catalase, (b) glutathione metabolizing enzymes such as glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and glucose 6-phosphate dehydrogenase (G6PD), (c) the low molecular weight antioxidants (GSH, vitamins E and C) and protein carbonyl content. RESULTS: The increased concentrations of MDA, conjugated diene and hydroperoxide (index of the lipid peroxidation) were controlled (P < 0.001) in the phycocyanin-pretreated group. At the outset, the low molecular weight antioxidants were appreciably increased (P < 0.001), whereas the tissue protein carbonyl concentration was decreased (P < 0.001), suggesting that phycocyanin provides protection to renal cell antioxidants. It was noticed that the activities of antioxidant enzymes and glutathione metabolizing enzymes were considerably stabilized in rats pretreated with phycocyanin. CONCLUSION: We suggest that phycocyanin protects the integrity of the renal cell by stabilizing the free radical mediated LPO and protein carbonyl, as well as low molecular weight antioxidants and antioxidant enzymes in renal cells. Thus, the present analysis reveals that the antioxidant nature of C-phycocyanin protects the renal cell against oxalate-induced injury and may be a nephroprotective agent.

Prophylactic role of phycocyanin: a study of oxalate mediated renal cell injury.

Oxalate induced renal calculi formation and the associated renal injury is thought to be caused by free radical mediated mechanisms. An in vivo model was used to investigate the effect of phycocyanin (from Spirulina platensis), a known antioxidant, against calcium oxalate urolithiasis. Male Wistar rats were divided into four groups. Hyperoxaluria was induced in two of these groups by intraperitoneal infusion of sodium oxalate (70 mg/kg) and a pretreatment of phycocyanin (100 mg/kg) as a single oral dosage was given, 1h prior to sodium oxalate infusion. An untreated control and drug control (phycocyanin alone) were also included in the study. We observed that phycocyanin significantly controlled the early biochemical changes in calcium oxalate stone formation. The antiurolithic nature of the drug was evaluated by the assessment of urinary risk factors and light microscopic observation of urinary crystals. Renal tubular damage as divulged by urinary marker enzymes (alkaline phosphatase, acid phosphatase and gamma-glutamyl transferase) and histopathological observations such as decreased tubulointerstitial, tubular dilatation and mononuclear inflammatory cells, indicated that renal damage was minimised in drug-pretreated group. Oxalate levels (P < 0.001) and lipid peroxidation (P < 0.001) in kidney tissue were significantly controlled by drug pretreatment, suggesting the ability of phycocyanin to quench the free radicals, thereby preventing the lipid peroxidation mediated tissue damage and oxalate entry. This accounts for the prevention of CaOx stones. Thus, the present analysis revealed the antioxidant and antiurolithic potential of phycocyanin thereby projecting it as a promising therapeutic agent against renal cell injury associated kidney stone formation.