HUVEC were incubated with mixtures of 50 μg/mL WT FI or mutants and C3b/125I-C3b Selleckchem BAY 80-6946 at 37°C. As positive control 20 μg/mL FH was added and in the negative control FI was omitted. The presence of cleavage products of C3b degradation was assessed by gradient SDS-PAGE (Fig. 7A). The intensity of the 68-kDa-cleavage product was calculated and presented as mean value from three independent experiments (Fig. 7B). The results demonstrate that endogenous membrane-bound MCP acts

as cofactor for FI-mediated cleavage of C3b. D501N mutant did not cleave C3b α′-chain, while P32A and A222G cleaved C3b as efficiently as the WT FI. In the presence of membrane-bound MCP as cofactor M120V, H165R and R299W degraded C3b α′-chain significantly more MK-8669 chemical structure efficiently than WT FI (Fig. 7B). We next tried to rationalize the functional consequences of several of the point mutations examined above in the context of the predicted three-dimensional (3D) structures of each domain of FI (Fig. 8). The homology modeling approach is described in detail in 34, although further details regarding the modeling of the FIMAC domain are given below. The models of the domains, FIMAC, CD5, LDLr1 and SP (Fig. 8) are presented separately because at present there are no reliable experimental data to suggest

how the domains are oriented in the full-length protein. The structural and functional consequences of the mutations are listed in Table 2. The residue Cys25 is located in the FIMAC domain and forms a disulfide bond with the adjacent Cys36 (Fig. 8). A mutation to

Phe destroys this stabilizing bond and further destabilizes by introducing steric clashes with the side chain of Cys36. It is likely that the Cys25 mutation imposes structural changes within the N-terminal region of this domain, possibly explaining why a decreased secretion of this mutant is observed experimentally. The Pro32 residue is fully Casein kinase 1 solvent exposed in a surface loop, at least in the isolated FIMAC domain (Fig. 8). Proline usually imposes greater conformational constraints on the polypeptide backbone than other amino acids and, in places where it can be tolerated, such as in loops and turns, proline makes a positive contribution to protein stability through entropic effects. In the present situation, while this mutation could slightly destabilize the domain, it could be structurally tolerated at this position. We found that the P32A mutant expressed as well as WT FI, and showed only slightly reduced function in degrading C4b and C3b in solution and only when C4BP and FH were used as cofactors. However, the P32A mutant showed substantially impaired ability to degrade C3b deposited on cell surfaces. A proline at position 32 could perturb interdomain contacts or form new interactions with a FI ligand when C3b is part of a deposited C3-convertase. The residue Met120 is located in the CD5 domain (Fig.

Accordingly, there was no recovery of FVIII activity 30 min after FVIII injection to the BM/FVIII, while FVIII RAD001 chemical structure recovery in BM/PBS was 0·69 ± 0·15 IU/ml (Supporting information

Fig. S1). In the case of BM/PBS mice, an anti-FVIII immune response developed with kinetics similar to that previously described;12 anti-FVIII IgG developed from the third FVIII administration and titres reached 767·6 ± 271·5 μg/ml after the fifth FVIII administration. In contrast, BM/FVIII mice developed negligible anti-FVIII IgG titres even 5 days after the fourth administration of FVIII (15 ± 19·4 μg/ml), compared with BM/PBS mice (179·5 ± 138 μg/ml, P < 0·01). In BM/FVIII mice, however, anti-FVIII IgG development initiated after the fifth injection of FVIII (103·3 ± 94 μg/ml) and reached 460 ± 278·2 μg/ml after a sixth FVIII administration. Similar results were obtained when inhibitory titres were measured in the serum of the mice using a Bethesda assay (Fig. 2b). Importantly, transfer of maternal anti-FVIII IgG influenced neither the total levels of circulating

IgG in the offspring (Fig. 2c), nor the capacity of the SCH727965 datasheet offspring to mount classical immune responses to an unrelated exogenous antigen such as OVA (Fig. 2d). We then analysed the effect of the transfer of maternal anti-FVIII IgG on FVIII-specific cellular immune responses. Splenocytes from BM/FVIII and BM/PBS mice administered five times with FVIII, were stimulated with FVIII in vitro. T cells from BM/FVIII and BM/PBS mice demonstrated identical capacities to proliferate in the presence of concanavalin A (Fig. 2e). In contrast, splenocytes from BM/FVIII mice marginally proliferated upon stimulation with FVIII compared

with splenocytes from BM/PBS mice; the ratios of stimulation indices being 1·63 ± 0·38 versus 3·09 ± 0·83, respectively (P < 0·05). Together, the data suggest that the transfer of anti-FVIII IgG from the mother to the progeny is associated with a reduced capacity to develop an anti-FVIII immune response. The transfer of maternal IgG to the offspring occurs during gestation through the placenta and during lactation through the intestinal epithelium.4 We investigated which of the two types of transfer is critical to impair the capacity of the progeny to develop an antigen-specific Tenofovir cost immune response. Mothers of BM/FVIII and BM/PBS mouse pups were interchanged at the time of birth so that some BM/PBS pups received anti-FVIII IgG during lactation (B/PBSM/FVIII) and some BM/FVIII pups did not receive antibodies from birth until the start of the FVIII immunization protocol (B/FVIIIM/PBS). In parallel, some BM/FVIII and BM/PBS pups were kept with their original mothers (referred to as B/FVIIIM/FVIII and B/PBSM/PBS, respectively). The pups were weaned at 5 weeks of age. At 8 weeks of age, B/FVIIIM/PBS mice did not have residual maternal anti-FVIII IgG, as assessed by ELISA (Fig.

No impact of HGG on the rate of transplant rejection was observed. The impact of treatment of HGG with IVIg was also presented. The authors would like to thank Meridian HealthComms Ltd for providing medical writing services. S. C. J. has received consultation and grant support from CSL Behring and Genentech-Roche. D. G. has received support for consulting, conferences and/or research from CSL Behring, One Lambda, Astellas and ROTRF.

“
“The immunoprophylactic and therapeutic potentials of root extracts of Withania somnifera chemotypes (NMITLI-118, NMITLI-101) and pure withanolide–withaferin A was investigated against Leishmania donovani infection in hamsters. The naive animals, fed orally with immunostimulatory doses of chemotypes 101R, 118R (10 and 3 mg/kg) and withaferin A (9 and 3 mg/kg) for five consecutive days and challenged

p38 MAPK phosphorylation with Leishmania parasites on day 6, were euthanized on days 30 and 45 p.c. for the assessment of parasite clearance, CDK inhibitor real-time analysis of mRNAs of Th1/Th2 cytokines (IFN-γ, IL-12, TNF-α, iNOS/IL-4, IL-10 and TGF-β), NO production, reactive oxygen species (ROS) generation, lymphocyte transformation test and antibody responses. By day 45 p.c., there was a significant increase in the mRNA expression of iNOS, IFN-γ, IL-12 and TNF-α but decrease in IL-4, IL-10 and TGF-β, an enhanced Leishmania-specific LTT response as well as ROS, NO and antileishmanial IgG2 levels in 101R-treated hamsters followed by 118R- and withaferin A-treated ones, respectively. When these chemotypes were given to L. donovani-infected hamsters at different doses, there was moderate therapeutic efficacy of chemotype 101R (~50%) at 30 mg/kg × 5 followed by the other two. The results established that

the 101R is the most potential chemotype and can be evaluated for combination therapy along with available antileishmanials. “
“Nontyphoidal Salmonellae commonly cause fatal bacteraemia in African children lacking anti-Salmonella antibodies. These are facultative intracellular bacteria capable Tangeritin of cell-free and intracellular survival within macrophages. To better understand the relationship between extracellular and intracellular infection in blood and general mechanisms of Ab-related protection against Salmonella, we used human blood and sera to measure kinetics of Ab and complement deposition, serum-mediated bactericidal killing and phagocytosis of invasive African Salmonella enterica serovar Typhimurium D23580. Binding of antibodies peaked by 30 s, but C3 deposition lagged behind, peaking after 2–4 min. C5b-9 deposition was undetectable until between 2 and 6 min and peaked after 10 min, after which time an increase in serum-mediated killing occurred. In contrast, intracellular, opsonized Salmonellae were readily detectable within 5 min. By 10 min, around half of monocytes and most neutrophils contained bacteria. The same kinetics of serum-mediated killing and phagocytosis were observed with S.

S. Environmental Protection Agency General Neurotoxicology Screening U.S. Environmental Protection Agency Delayed Neurotoxicity Screening U.S. Environmental Protection Agency Developmental Neurotoxicity Screening U.S. Food and Drug Administration General Neurotoxicology Screening U.S. Food and Drug Administration Developmental Neurotoxicity Screening References “
“Rosette-forming glioneuronal SB203580 research buy tumors (RGNT) of the fourth ventricle are rare mixed glio-neuronal tumors included in the revised WHO classification of CNS tumors and show histopathological features similar to pilocytic astrocytomas. To evaluate at molecular level potential affinities

between these tumors, we investigated a case of RGNT, arising in the cerebellum of a young patient, for the presence of transcriptional products originating from the KIAA1549-BRAF fusion. However, the analysis did not show any fusion. Further studies in larger RGNT case series LDE225 clinical trial are needed in order to demonstrate the possible presence of KIAA1549-BRAF fusion and better delineate its relationship with pilocytic astrocytomas. “
“We report a rare case of ependymoma with vacuolar features, signet cells, pigmentation and numerous Rosenthal fibers arising in the fourth ventricle of a 35-year-old woman. The tumor was composed of cells with cytoplasmic vacuoles, signet cells and clear cells. The clear

cells were compactly arranged resembling oligodendroglioma. Pseudovascular and ependymal rosettes were observed only in focal areas. Additionally, some tumor cells contained

brown cytoplasmic pigment, which was histochemically compatible with lipofuscin and neuromelanin. On immunohistochemical examination, the tumor cells were positive for S100, glial fibrillary acidic protein and vimentin, and negative for synaptophysin, cytokeratin, neurofilament and HMB45. Epithelial membrane antigen staining showed dot-like and small vesicular reactivity. The case is presented to increase familiarity with these extraordinary variants of ependymoma. “
“To investigate the clinicopathological features of anaplastic astrocytoma (AA) with abundant Rosenthal fibers (RFs), this study assessed four cases of AA (elderly patients; age ≥70 years). Bay 11-7085 Histologically, these tumors were composed of diffusely infiltrating astrocytomas with brightly eosinophilic cytoplasmic granules or cork-screw or beaded bundles. Tumor cells showed pleomorphism, bizarre giant cells, and mitotic activity, but no necrosis. The cytoplasmic granules showed negativity on PAS staining. Immunohistochemically, the tumor cells with cytoplasmic granular cells showed a positive reaction for GFAP. The cytoplasmic eosinophilic granules or bundles were positive for αB-crystallin, ubiquitin and HSP27. In addition, tumor cells showed strong cytoplasmic positivity for isocitrate dehydrogenase 1 (IDH1)-R132H protein in all cases.

Here, we will argue that the requirement for a stable MHC interaction is one of those “other” factors. It is generally recognized that GSK-3 inhibitor the requirement for binding

and presentation by MHC-I molecules is by far the most selective event of antigen processing and presentation [[6, 22-24]]. When searching for CD8+ T-cell epitopes, an affinity better than 500 nM (termed a good binder) is commonly used as a threshold to select candidate immunogenic peptides [[25]]. Sette and colleagues recently estimated that “the vast majority of epitopes (85%) bound their restricting MHC-I with an affinity of 500 nM or better, and most (75%) bound with an affinity of 100 nM or better” [[6]]. Unfortunately, this criterion leads to the inclusion of many nonimmunogenic peptides (i.e. false positives). Others and

we have observed that only some 10–20% of pathogen-derived peptides, which bind to MHC-I with an experimentally verified affinity of 500 nM, or better, are subsequently found to be immunogenic [[6, 25, 26]]. Testing the immunogenicity of all predicted immunogenic epitopes is currently a very slow, costly process, and any computational T-cell epitope discovery process would benefit from a better and more quantitative understanding of antigen processing and presentation. It has been suggested that the stability of pMHC complex correlates with immunogenicity (both for MHC-I [[1, 27-32]], and for MHC-II ABT-199 research buy [[2, 33]]); and it has even been suggested that stability correlates better with immunogenicity than affinity of peptide interaction

with MHC-I [[34-37]] and MHC-II [[38]]. Common Oxymatrine to all these reports is that the experimental data are limited to a few epitopes. Here, we have examined the stability of 739 peptides that bind to HLA-A*02:01 with an affinity of about 1000 nM or better. We found that the rate of dissociation at 37°C varied from a half-life of over 40 h to one of less than 0.1 h. To neutralize the effect of affinity, affinity-balanced pairs of known versus “not-known-to-be” immunogens restricted to different HLA alleles (A*01:01, A*02:01, B*07:02, and B*35:01) were extracted and analyzed biochemically. We found a highly significant difference in the stability of immunogens compared to “not-known-to-be” immunogens for three of the four HLA class I molecules examined. In parallel studies of the immunogenicity of HIV-derived epitopes restricted to B*57:02, B*57:03, B*58:01, B*07:02, B*42:01, and B*42:02, we have found that stability is a better discriminator of immunogenicity than affinity is (Kløverpris et al., manuscripts in preparation). Thus, the proposition that stability is a better indicator of immunogenicity can be extended to a wide range of HLA class I molecules. We were, however, concerned that the underlying data set was not representative of an unbiased epitope discovery process, since many reported CTL epitopes have been discovered using simple rule-based predictions of high-affinity binding to MHC-I.

The direct transport of the diuretic drugs via these oocyte experiments were quantified selleck chemicals llc by robust LC/MS-MS methods. Results: Loop diuretics (bumetanide, ethacrynic acid and furosemide), thiazide diuretics (chlorothiazide, hydrochlorothiazide and trichlormethazide), carbonic anhydrase inhibitors (acetazolamide and methazolamide) and amiloride, a potassium-sparing diuretic that acts on epithelial sodium channel (ENaC), but not spironolactone, a potassium-sparing

diuretics with mineralocorticoid receptor antagonist, were significantly transported into oocytes expressing OAT1, OAT3 and NPT4. It is interesting that acetazolamide, amiloride and methazolamide click here are transported by NPT4 even though they did not show significant inhibition on NPT4-mediated PAH or urate transport. Conclusion: To our knowledge, these findings are the first report which illustrate that the basolateral organic anion transporters OAT1 and OAT3 and

an apical voltage driven-organic anion transporter NPT4 are directly involved in trans-cellular secretion of various diuretic drugs across renal proximal tubular cells. The interaction of thiazides and loop diuretics on NPT4 may help to explain the known clinical observations pertaining to “Diuretics-induced hyperuricemia”. MAJUMDAR ARGHYA1,2, JAIN ADITI2 1Head, Dept of Nephrology, AMRI Hospitals, Kolkata, India; 2Post graduate trainee, AMRI Hospitals, Kolkata, India Introduction: To study the effectiveness of microalbuminuria (MA), a marker of endothelial dysfunction, in delineating sepsis from SIRS, the role of VEGF/ sFLT in its pathophysiology and its clinical implications. Methods: Setting:

Multi-specialty intensive care unit in a tertiary hospital (AMRI) in Kolkata Study Duration: 1 year Study Design: Prospective observational study. Inclusion Criteria: Adult patients (>18 yrs age) with features of systemic inflammatory Fossariinae response syndrome/sepsis admitted to ICU. Exclusion criteria: Patients less than 18 yrs age, brought in from other health facilities or transferred from the wards after more than 24 hours of in hospital stay, post- surgical pts, those anuric (for the first 6 hours of admission), with macroscopic hematuria, hemoglobinuria, pregnant or menstruating women, patients with neoplasm, known cases of CKD and macroalbuminuria. Methods: Urine MA and serum VEGF and sFLT levels were measured on admission and after 24 hours in all critically ill patients with SIRS. Clinical data was collated. Results: After screening 184 patients with SIRS, 40 were studied- mean age 57 years, 65% male,72.5% having been admitted to the ICU from home, 76.7% having SIRS due to sepsis. The average APACHE IV and APS score in the groups with SIRS due to sepsis and without and the disease duration were similar.

In addition, many spheroids, which are known early ALS changes in the PD-0332991 cell line anterior horn [12], were observed in the spinal anterior horn, and these findings were not described in the previously reported FALS cases with TARDBP mutations. Furthermore, TDP-43 pathology was rarely detected in the LMN of the present case whereas widespread TDP-43 pathology in the previously reported FALS cases with TARDBP mutations (Table 2). Such histopathological features in our case seem to be suggesting the possibility that FALS with p.N352S mutation in TARDBP might have neuropathological differences at the point of distribution and degree of neurodegenerative lesions compared with autopsy-confirmed FALS cases

with other mutations in TARDBP, although further case accumulation and analyses are needed. p.N352S mutation in TARDBP have been predicted to increase TDP-43 phosphorylation, resulting in TDP-43 accumulation [5]. However,

pTDP-43-immunoreactive deposits were rare in our case, suggesting that this mutation in TARDBP is less capable of causing pTDP-43 aggregation, resulting in slight to mild neuronal loss with restricted lesional distribution. Thus, further studies, including transgenic animal studies, are needed to elucidate the discrepancies between the extent LBH589 of TDP-43 pathology and the histopathological lesional distribution of FALS among different mutations. In conclusion, we described the clinicopathological characteristics of a FALS case with p.N352S mutation in TARDBP. Further clinicopathological analyses are needed to more clearly identify the clinicopathological features of FALS with p.N352S mutation in TARDBP. We sincerely thank Mr Mitsuhiro Ikeda, Mrs Yoshie Ishizaka, Mrs Nao Hiraishi and Mrs Yoko Suzuki from the Tokyo Metropolitan Neurological Hospital for their excellent technical assistance. “
“Adult onset leukodystrophy with neuroaxonal spheroids is an uncommon cause of dementia.

Both hereditary Interleukin-2 receptor (autosomal dominant) and sporadic cases have been described. A 41-year-old African woman presented with inappropriate behavior and personality change consistent with frontal lobe dysfunction. MRI demonstrated diffuse frontoparietal white matter signal abnormality and volume loss, as well as focal enhancing white matter lesions, while CT scan showed white matter calcifications. She had been gradually deteriorating over the last 5 years, diagnosed as having progressive demyelinating illness. She died of recurrent chest infections. There was no familial history. The brain showed prominent symmetrical white matter changes with greyish discolorization mainly affecting the frontal and parietal lobes, with less involvement of the temporal lobe and only mildly affecting the occipital white matter. Histology revealed deep white matter atrophy with many neuroaxonal spheroids labelled by neurofilament and β-amyloid precursor protein.

The study was approved by the ethics committee of Pasteur Institute of Iran. The four strains along with the reference strain (RS) of L. major (MRHO/IR/75/ER) as a control, were used for inoculation of BALB/c mice. Fifty thousand stationary

phase promastigotes were inoculated in the right foot pad of BALB/c mice. Parasite was grown in RPMI 1640 media, supplemented with 2 mM L-glutamine, 10% foetal bovine serum, 100 U/mL penicillin and 100 μg streptomycin and then harvested and washed with Phosphate buffered saline (PBS) by centrifugation at 3000 rpm for 30 min. Species of the strains were characterized by isoenzyme electrophoresis and PCR as L. major. Genetic heterogeneity of the four strains was analysed by single-strand conformation polymorphism Selleck Daporinad (SSCP).The internal transcribed spacer 1 (ITS1) was amplified by primer pairs L5.8S (5′- TGATACCACTTATCG-CACTT -3′) and LITSR (5′ – CTGGATCATTTTCCGATG -3′) as described

previously [15]. Amplification reactions were carried out in volumes of 25 μL: 60 ng DNA was mixed with a PCR mixture containing 200 μM dNTPs mix, 1.5 mM MgCl2, 1 U Taq polymerase and 10 pmol of each primer. The amplification of samples was performed at: 95°C for 5 min for initial denaturing followed by 35 cycles consisting of denaturation at 95ºC for Cabozantinib 40 s, annealing at 60ºC for 40 s and extension at 72ºC for 1 min. Final extension was followed at 72ºC for 7 min. PCR products were analysed on 1.5% agarose gel, and the bands Temsirolimus were visualized by ethidium bromide staining [16]. Single-strand conformation polymorphism was performed by denaturing the double-strand DNA products as described (15), with a few modifications. Briefly, 4 μL of PCR products were mixed with 6 μL denaturing buffer (95% formamide, 10 mm NaOH, 0·25% bromophenol blue, 0·25% Xylene) and 4 μL loading buffer (40% Sucrose, 0·25% bromophenol blue,

0·25% Xylene). After heating at 98ºC, the mixture was immediately frozen in liquid nitrogen for 15 min. The samples were loaded then on 5.5% polyacrylamide gel and silver stained. For assessment of cytokine mRNA, 35 mice in each group were inoculated with five strains (175 mice), and cytokine transcripts were analysed in each time point of 3, 16, 40 h, 1 week, 3, 5 and 8 weeks post-infection (five mice for each time point). One group containing five un-infected mice were used as a control. Parasite load was measured by inoculation of four mice in each group with five strains (20 mice in total) and after 8 weeks, parasite burdens were determined in LN of each mouse. Parasite load was estimated at 8 weeks post-infection.

Meanwhile, we found aberrant expression of some proteins associated with oxidative stress, nitric oxide and the ubiquitin-proteasome system. AGEs, a marker for oxidative stress, which was over-expressed in abnormal fibres as

reported previously [18], can promote the abnormal oxidation of aggregated proteins. Over-expression of eNOS, associated with reduction of nitric oxide, may result in protein nitration and motivate toxic reactivity of aggregated proteins [18]. Mutant ubiquitin is a kind of misreading ubiquitin. Over-expression of mutant ubiquitin in the abnormal fibres indicated that the mutant desmin can impair the proteolytic function of the ubiquitin-proteasome system. The over-expression of p62 could be a response to disturbance of the ubiquitin-mediated find more process [19]. Up to now, a total of 44 mutations responsible for desminopathy have been identified in the world. Many mutations were clustered in the helix 2B domain of desmin, and formed the hotspot region in Caucasian populations [8,34]. However, it is interesting that so many de

novo mutations (six of seven mutations) of the desmin gene were identified in the current series of patients. A different genetic background in affected patients is likely to further modify the clinical manifestations of disease in different selleck monoclonal antibody populations. The novel S12F mutation located in the first site of a highly conserved nonpeptide motif (SSYRRTFGG) in the head domain of desmin is shared by other human intermediate filaments and conserved in the evolutionary tree. The loss of the Ser12 residue might alter the phosphorylation in the head domain, see more thus affecting desmin filament assembly and disassembly [22,35]. The four other mutations in helix 1A, 2A and 2B of the rod domain affected the mosaic arrangement of hydrophilic and

hydrophobic amino acids in the conserved heptad repeat. The changes were likely to decrease the local flexibility of a coiled-coil rod domain, thus obstructing the proper assembly of desmin intermediate filaments [36]. The T445A and E457V mutations were located in the highly conserved β-turn motif of the tail domain, which seems to be essential for inter-protofibrillar stability and width control, and thus interfered with the binding of desmin filaments to other proteins that are cofactors of the cytoskeleton, or parts of muscle-specific signalling cascades [37]. Since all novel mutations were distributed in several domains of desmin, it seemed unlikely that Chinese patients belonged to a distinctive group of desminopathy. Our functional studies provided compelling evidence that six mutants severely affected the ability of desmin to produce a filamentous network in a desmin-negative cell line.

All four groups were killed 16 h postoperative with an overdose of a general anaesthetic (thiopental sodium, 50 mg/kg). The lungs and kidneys were removed quickly from all the rats and washed in ice-cold saline. Half the tissues were transferred to a biochemistry laboratory to be kept at −80°C click here for biochemical analyses, and the other half of the tissues were fixed in 10% formalin solution for histopathological analyses. After macroscopic analyses, activities of superoxide dismutase (SOD) and myeloperoxidase (MPO) and amounts of lipid peroxidase (LPO) and glutathione (GSH) enzymes in the rat lung and kidney tissues were determined. To prepare the tissue

homogenates, the tissues were ground with liquid nitrogen in a mortar. The ground tissues (0·5 g each) were then treated with 4·5 ml of the appropriate buffer.

The mixtures were homogenized on ice using an Ultra-Turrax Homogenizer for 15 min. The homogenates were filtered and centrifuged, using a refrigerated centrifuge at 4°C. These supernatants were then used to determine enzymatic activity. All assays were performed at room temperature in triplicate. Measurements were made according to the method of Sun et al. [45]. SOD estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which react with nitroblue tetrazolium (NTB) to form formazan dye. SOD activity was then measured at 560 nm by the degree of inhibition of this reaction and was see more expressed as mmol/min/mg/tissue. MPO activity was measured according to the modified method of Bradley

et al. [46]. The homogenized samples were frozen and thawed three times and then centrifuged at 1500 g for 10 min at 4°C. MPO activity was determined by adding 100 µl of the supernatant to 1·9 ml of 10 mmol/l phosphate buffer (pH 6·0) and 1 ml of 1·5 mmol/l o-dianisidine hydrochloride containing 0·0005% (wt/vol) hydrogen peroxide. The changes in each sample’s absorbance at 450 nm were recorded below on a UV–vis spectrophotometer. MPO activity in all tissues was expressed as µmol/min/mg/tissue. LPO in the tissues was determined by estimating the level of malondialdehyde (MDA) using the thiobarbituric acid test [47]. The rat tissues were excised promptly and rinsed with cold saline. To minimize the possibility of the interference of haemoglobin with the free radicals, any blood adhering to the mucosa was removed carefully. The tissues were weighed and homogenized in 10 ml of 100 g/l KCl. The homogenate (0·5 ml) was added to a solution containing 0·2 ml of 80 g/l sodium lauryl sulphate, 1·5 ml of 200 g/l acetic acid, 1·5 ml of 8 g/l 2-thiobarbiturate and 0·3 ml of distilled water. The mixture was incubated at 98°C for 1 h. After the mixture cooled, 5 ml of n-butanol : pyridine (15 : l) was added. The mixture was centrifuged for 30 min at 896 g.