Repetitive nasal allergen challenge in allergic rhinitis: Priming and Th2-type inflammation but no evidence of remodelling.

BACKGROUND: Local tissue eosinophilia and Th2 cytokines are characteristic features of seasonal allergic rhinitis. Airway remodelling is a feature of asthma whereas evidence for remodelling in allergic rhinitis (AR) is conflicting. OBJECTIVE: By use of a novel human repetitive nasal allergen challenge (RAC) model, we evaluated the relationship between allergic inflammation and features of remodelling in AR. METHODS: Twelve patients with moderate-severe AR underwent 5 alternate day challenges with diluent which after 4 weeks were followed by 5 alternate day challenges with grass pollen extract. Nasal symptoms, Th1/Th2 cytokines in nasal secretion and serum were evaluated. Nasal biopsies were taken 24 hours after the 1st and 5th challenges with diluent and with allergen. Sixteen healthy controls underwent a single challenge with diluent and with allergen. Using immunohistochemistry, epithelial and submucosal inflammatory cells and remodelling markers were evaluated by computed image analysis. RESULTS: There was an increase in early and late-phase symptoms after every allergen challenge compared to diluent (both P < .05) with evidence of both clinical and immunological priming. Nasal tissue eosinophils and IL-5 in nasal secretion increased significantly after RAC compared to corresponding diluent challenges (P < .01, P = .01, respectively). There was a correlation between submucosal mast cells and the early-phase clinical response (r = 0.79, P = .007) and an association between epithelial eosinophils and IL-5 concentrations in nasal secretion (r = 0.69, P = .06) in allergic rhinitis. No differences were observed after RAC with regard to epithelial integrity, reticular basement membrane thickness, glandular area, expression of markers of activation of airway remodelling including α-SMA, HSP-47, extracellular matrix (MMP7, 9 and TIMP-1), angiogenesis and lymphangiogenesis for AR compared with healthy controls. CONCLUSION: Novel repetitive nasal allergen challenge in participants with severe persistent seasonal allergic rhinitis resulted in tissue eosinophilia and increases in IL-5 but no structural changes. Our data support no link between robust Th2-inflammation and development of airway remodelling in AR.

HPLC/DAD, Antibacterial and Antioxidant Activities of Plectranthus Species (Lamiaceae) Combined with the Chemometric Calculations.

The increase in antibiotic resistance and the emergence of new bacterial infections have intensified the research for natural products from plants with associated therapy. This study aimed to verify the antibacterial and antioxidant activity of crude extracts of the genus Plectranthus species, being the first report on the modulation of aminoglycosides antibiotic activity by Plectranthus amboinicus extracts. The chemical composition was obtained by chemical prospecting and High-Performance Liquid Chromatography with diode arrangement detector (HPLC/DAD). The antibacterial activities of the extracts alone or in association with aminoglycosides were analyzed using the microdilution test. The antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging. The phytochemical prospection allowed the flavonoids, saponins, tannins and triterpenoids to be identified. Quercetin, rutin, gallic acid, chlorogenic acid, caffeic acid, catechin, kaempferol, glycosylated kaempferol, quercitrin, and isoquercitrin were identified and quantified. The principal component analysis (PCA) observed the influence of flavonoids and phenolic acids from Plectranthus species on studied activities. Phytochemical tests with the extracts indicated, especially, the presence of flavonoids, confirmed by quantitative analysis by HPLC. The results revealed antibacterial activities, and synergistic effects combined with aminoglycosides, as well as antioxidant potential, especially for P. ornatus species, with IC50 of 32.21 µg/mL. Multivariate analyzes show that the inclusion of data from the antioxidant and antibacterial activity suggests that the antioxidant effect of these species presents a significant contribution to the synergistic effect of phytoconstituents, especially based on the flavonoid contents. The results of this study suggest the antibacterial activity of Plectranthus extracts, as well as their potential in modifying the resistance of the analyzed aminoglycosides.

A Novel Quantitative Approach to Staging and Assessing Recovery from Type 1 Diabetes Mellitus: The Type 1 Diabetes Mellitus Metabolic Recovery Index.

Discovery of insulin in 1921 changed the lives of patients with type 1 diabetes (T1DM) forever. What had been a death sentence became a manageable, albeit chronic, disease. Insulin did not cure the disease, as it did not address the actual disease process, but instead treated its sequelae, namely elevated blood sugars. Importantly, insulin administration fails to ensure normoglycaemia. Even with the most sophisticated 'near closed-loop' methods, glucose homeostasis is not restored to normal. T1DM patients face complications, both short-term, such as hypo- and hyperglycaemia, and long-term, with increased glycosylation of proteins leading to eye, kidney, nervous system and other sequelae. These complications are associated with significant morbidity and mortality even after intensive insulin treatment. Nearly 100 years after the discovery of insulin, we continue to face the challenge of addressing the disease process itself, in order to fundamentally improve the life of these patients. There are major efforts to achieve just that: to completely arrest the autoimmune process destroying the insulin-producing cells in the pancreas, or at least significantly slow the process to blunt and delay short- and long-term complications. The aim of this Communication is to propose a novel assessment tool that would serve as a quantitative outcome measure by which therapies, short of clinical cure, may be compared and their true benefit to the treatment of diabetes assessed.

Severe Persistent Allergic Rhinitis. Inflammation but No Histologic Features of Structural Upper Airway Remodeling.

RATIONALE: Increases in airway smooth muscle, extracellular matrix, and vascularity are prominent features of airway remodeling in asthma, whereas the extent of such remodeling in patients with persistent allergic rhinitis (PAR) is unknown. OBJECTIVES: To test the hypothesis that upper airway remodeling is a feature of PAR. METHODS: Total nasal symptoms scores, nasal biopsies, and Th1 and Th2 cytokines from nasal lavage were assessed in subjects with severe PAR (n = 46) and healthy control subjects (n = 19). Angiolymphangiogenesis was examined using immunohistochemistry staining against CD31 (vascular endothelial cells), vascular endothelial growth factor-A, and D2-40 (lymphatic endothelial cells). Collagen and extracellular matrix proteins, such as heat shock protein-47 (markers of collagen synthesis), matrix metalloproteinase-9, and tissue inhibitor metalloproteinase-1, and α-smooth muscle actin (myofibroblasts) were evaluated as markers of activation of upper airway remodeling using image analysis, together with reticular basement membrane thickness, mucus gland area, collagen area, and submucosal effector inflammatory cells. MEASUREMENTS AND MAIN RESULTS: Total nasal symptoms scores, visual analog scale, and total quality of life were significantly higher in PAR compared with healthy control subjects (P < 0.0001). Nasal lavage cytokine levels of IL-4 (P < 0.01), IL-5, and IL-13 (P < 0.001, respectively) were significantly higher in PAR compared with healthy control subjects. In addition there was an increase in submucosal eosinophils (P = 0.06). No statistical difference in terms of angiogenesis, lymphangiogenesis, deposition of extracellular matrix, collagen markers, reticular basement membrane thickness, or glandular percentage area was observed between PAR and healthy control subjects. CONCLUSIONS: Our data suggest that tissue remodeling is not a feature of PAR and argues that in contrast to asthma, targeting remodeling in allergic rhinitis may not be appropriate as a therapeutic approach.

Morphological, histochemical and immunohistochemical characterization of secretory production of the ciliary glands in the porcine eyelid.

In addition to performing general histology and cytology of the ciliary glands of the miniature pig, we studied the localization of glycoconjugates and beta-defensins in these glands with the use of carbohydrate histochemical and immunohistochemical methods. The secretory cells of the glands were equipped with non-homogeneous secretory granules, a well-developed Golgi apparatus and rough endoplasmic reticulum. The secretory epithelium and luminal secretion of the glands contained large amounts of neutral and acidic glycoconjugates with various saccharide residues (alpha-L-Fuc, beta-D-Gal, alpha-D-GalNAc and sialic acid). The sebaceous glands and tarsal glands also exhibited positive reactions to most of the histochemical methods. Additionally, the antimicrobial peptide group of beta-defensins was demonstrated to be products of the ciliary glands, as well as the sebaceous glands and tarsal glands. The results obtained are discussed with regard to the specific function of the ciliary glandular secretions. These secretory products may be related to the moistening and general protection of the skin surface of the eyelid and ocular surface.

Carbamoyl-phosphate synthetase II in kinetoplastids.

Genes for carbamoyl-phosphate synthetase II (CPS II), the first enzyme of de novo pyrimidine biosynthesis, were cloned from kinetoplastids, Trypanosoma cruzi and Leishmania mexicana. T. cruzi CPS II gene encodes a protein of 1524 amino acids that encompasses the glutaminase and CPS domains, but incorporates neither aspartate carbamoyltransferase nor dihydroorotase. The residue corresponding to lysine 993 of Escherichia coli CPS, a residue that characterizes the CPS inhibited by UMP and that is replaced by tryptophan in those inhibited by UTP, is in kinetoplastids a hydrophilic glutamine, in line with the preferential inhibition by UDP of kinetoplastid CPS II.

Inhibition of Fas-mediated apoptosis by Trypanosoma cruzi infection.

Trypanosoma cruzi-infected and normal control mammalian cells were subjected to analysis of Fas-mediated apoptosis stimulated by an agonistic anti-Fas monoclonal antibody. The infected cells showed markedly hampered apoptotic changes in nuclear morphology, phosphatidylethanolamine translocation from the inside to the outside of the plasma membrane, and DNA fragmentation into multiples of 180 bp, relative to normal control cells. Upstream of these morphological and biochemical consequences, the caspase-3 activity was elevated by the Fas stimulation in a significantly greater proportion of intact control cells, but at a highly reduced rate of infected cells. The rapid elevation of caspase-8 activity in control, apoptotic cells was completely inhibited in infected cells. In an examination of the specificity of other stimulants, X-ray radiation or chemicals such as hydrogen peroxide, colchicine or etoposide did not cause significant differences in apoptotic rates between control and infected cells; tumor necrosis factor-alpha, however, induced a high rate of apoptosis in control cells, with an extremely lowered rate in infected cells. This study demonstrates, for the first time, that T. cruzi infection inhibits one of the earliest steps of death receptor-mediated apoptosis, an effect that most probably involves the inhibition of caspase-8. Differential apoptotic responses in cells infected with T. cruzi and other intracellular parasites are discussed.

Novel organization and sequences of five genes encoding all six enzymes for de novo pyrimidine biosynthesis in Trypanosoma cruzi.

A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas' disease, was sequenced. It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6-5, encoding all six enzymes involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, dihydroorotate dehydrogenase, and orotidine-5'-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively. The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6-5, pyr2, and pyr4 from the 5' terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb. The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences. The last two enzymes encoded by a single gene, pyr6-5, are covalently linked in the order opposite to mammalian pyr5-6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus. The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes. We conclude that the T. cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1-3-2, pyr4, and pyr5-6, for UMP synthesis. Peculiarities in the trypanosomatid pyr6-5 gene product are discussed.

Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes.

The de-novo pyrimidine biosynthetic pathway involves six enzymes, in order from the first to the sixth step, carbamoyl-phosphate synthetase II (CPS II) comprising glutamine amidotransferase (GAT) and carbamoyl-phosphate synthetase (CPS) domains or subunits, aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase (DHOD), orotate phosphoribosyltransferase (OPRT), and orotidine-5'-monophosphate decarboxylase (OMPDC). In contrast with reports on molecular evolution of the individual enzymes, we attempted to draw an evolutionary picture of the whole pathway using the protein phylogeny. We demonstrate highly mosaic organizations of the pyrimidine biosynthetic pathway in eukaryotes. During evolution of the eukaryotic pathway, plants and fungi (or their ancestors) in particular may have secondarily acquired the characteristic enzymes. This is consistent with the fact that the organization of plant enzymes is highly chimeric: (1) two subunits of CPS II, GAT and CPS, cluster with a clade including cyanobacteria and red algal chloroplasts, (2) ACT not with a cyanobacterium, Synechocystis spp., irrespective of its putative signal sequence targeting into chloroplasts, and (3) DHO with a clade of proteobacteria. In fungi, DHO and OPRT cluster respectively with the corresponding proteobacterial counterparts. The phylogenetic analyses of DHOD and OMPDC also support the implications of the mosaic pyrimidine biosynthetic pathway in eukaryotes. The potential importance of the horizontal gene transfer(s) and endosymbiosis in establishing the mosaic pathway is discussed.

Cloning and functional expression of Rpn1, a regulatory-particle non-ATPase subunit 1, of proteasome from Trypanosoma cruzi.

Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles. We have cloned the gene RPN1 encoding Rpnl (regulatory-particle non-ATPase subunit 1), one of the largest subunits of proteasome, from Trypanosoma cruzi. It contains 2712 bp and encodes 904 amino acid residues with a calculated molecular mass of 98.2 kDa and an isoelectric point of 5.2. The predicted amino acid sequence of the trypanosomatid Rpn1 shares 39.0 and 32.0% overall identities with human Rpn1 and Saccharomyces cerevisiae Nas1 (non-ATPase subunit 1), an Rpn1 homolog, respectively, while the sequence identities among T. cruzi, Plasmodium falciparum, and Entamoeba histolytica Rpnl are approximately 30%. T. cruzi Rpn1 contains nine repeats of about 36 amino acid residues conserved in Rpn1s from various organisms. T. cruzi RPN1 is located on the 2300- and 1900-kb chromosomal DNA, displays a putative allelic variation as RPN1-1 and RPN1-2 with 98.8% identity between these two putative gene products, and is transcribed from both alleles at a comparable level throughout the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. The expression of the trypanosomatid Rpnl in the temperature-sensitive nas1 yeast mutant rescued the growth defect at the restrictive temperature, indicating that Rpn1 functions as a Nas1 and probably assembles into the 19S regulatory particle of the yeast 26S proteasome.

Duplication of part of 9q due to maternal 12;9 inverted insertion associated with pyloric stenosis.

We report on a 9-month-old boy who had duplication of the long arm of chromosome 9 [46,XY, -12, +der(12) inv ins (12;9)(p13;q32q13)mat.]. The clinical manifestations of the patient were different from those seen in distal 9q duplication. Pyloric stenosis appears to be common in cases with proximal 9q duplications.

Interstitial duplication 8q22-q24: report of a case proven by FISH with mapped cosmid probes.

We report on a 6-month-old malformed female infant with a de novo interstitial duplication of an 8q22-q24 segment. She had an excess dark-band on the 8q distal region by GTG-banded chromosome analysis, which was likely to be 8q23. We performed FISH analysis using cosmid probes mapped to 8q23 and proved that the patient had an 8q duplication including the 8q23 region.

Light and electron microscopic investigation of the process of healing of the naevus of Ota by Q-switched alexandrite laser irradiation.

Melanocytes in the naevus of Ota were destroyed by irradiation using the Q-switched alexandrite laser. This laser is highly selective and highly absorbed by melanosomes. Other cells and tissue components of the dermis remained almost intact. Melanosomes were vaporized or fragmented to subelectron microscopical size, or degenerated. If the irradiated energy was sufficient, melanocytes vanished and large vacuoles several times the size of dermal melanocytes formed at the sites. If it was too weak, dermal melanocytes were also vaporized, but vacuoles formed within them. Nuclei were no longer discernible. Following irradiation macrophages infiltrated the irradiated areas and scavenged degenerated melanosomes and cellular debris. Thus, discoloration of the skin was markedly reduced. Although a few melanocytes and melanophages remained, pigmentation cleared to a satisfactory level. Melanocytes and keratinocytes were also injured in the epidermis; however, the epidermis recovered completely. No scarring was observed.

Electron microscopic examination of oligocilia in naevus of Ota.

The structure of the cilia present in dermal melanocytes of 14 patients with naevus of Ota was examined by electron microscopy. Cilia and basal bodies were found in 10 and 9 lesions, and in 39 and 18 dermal melanocytes, respectively. In each case, 1-12 cells with a single cilium or multiple cilia were observed. In a total of 3 dermal melanocytes from 2 cases, two cilia per cell were observed. The cilia contained 7, 6, 5 and 4 pairs of doublet microtubules in the periphery and no central microtubule. Another pattern with several pairs of doublet microtubules in the periphery and one or two centrally located doublet microtubules were also observed. The latter were not bona fide central microtubules but one and two doublets, which seemed to be displaced to the centre from the periphery of the cilium.

Drug extrusion in Corynebacterium glutamicum.

We selected a mutant of Corynebacterium glutamicum, EBR, which can grow in a medium containing cytotoxic ethidium bromide (EtBr) at a high concentration of 100 microM. The resistance to EtBr in the mutant was reversed by 2 microM reserpine, a potent inhibitor of mammalian p-glycoprotein and bacterial multidrug resistance (MDR) transporter, whereas reserpine alone had a minimal effect on cell growth. The mutant showed a much higher efflux rate of EtBr than wild-type cells, and the efflux was completely inhibited by 2 microM reserpine. In addition to reserpine, structurally unrelated chemicals such as quinidine, trifluorperazine, tetraphenylarsonium chloride, chlorpromazine and quinine inhibit the EtBr efflux, revealing that the putative efflux system(s) can recognize a variety of chemicals. The efflux activity was correlated with the membrane potential but not the intracellular ATP contents. We, therefore, concluded that the EtBr resistance may be involved by proton-motive-force driven multidrug efflux system(s).

A comparison of the antischistosomal effect of levo- and dextro-praziquantel on Schistosoma japonicum and S. mansoni in mice.

Praziquantel (PZQ) is a racemic compound composed of equal proportions of its optical isomers, levo- and dextro-PZQ. The efficacy of these compounds was compared with that of PZQ in mice infected with Schistosoma japonicum or S. mansoni. Mice were given 50, 2 x 50 (on consecutive days), 500, or 2 x 250 mg/kg of each compound orally 5 weeks after infection. Significant reduction of worm recovery was observed in S. japonicum infection 30 days after treatment with 2 x 50, 500, or 2 x 250 mg/kg of levo-PZQ, whereas no therapeutic effect was demonstrated with dextro-PZQ. Percent reduction in worm burden in mice treated with levo-PZQ was significantly higher than in those with PZQ at a dosage of 2 x 50 mg/kg (67.9% vs. 34.5%). Neither eggs in feces nor miracidial hatching of eggs from the livers and intestines were observed in mice treated with levo-PZQ. In S. mansoni infection, levo-PZQ showed no significant schistosomicidal effect compared with PZQ and dextro-PZQ, although there was reduction in egg counts.

Schistosoma japonicum and S. mansoni: ultrastructural damage in the tegument and reproductive organs after treatment with levo- and dextro-praziquantel.

Ultrastructural observations were made of changes in the tegument and reproductive organs of Schistosoma japonicum and S. mansoni from ICR mice after treatment with praziquantel (PZQ), levo-PZQ, and dextro-PZQ at a single oral dose of 500 mg/kg body weight. No marked difference in types and extent of lesions of the tegument of S. japonicum was found between the compounds regardless of the time of worm recovery after treatment. This was equally true of S. mansoni. Degeneration of the testis, ovary, and vitelline gland of S. japonicum was more prominent in worms administered PZQ and levo-PZQ than in those receiving dextro-PZQ. In S. mansoni, extensive regression of the reproductive organs was observed in male and female worms treated with PZQ and dextro-PZQ, while no serious damage was seen in worms treated with levo-PZQ.

Serologic and ultrasonographic parameters of praziquantel treatment of hepatic fibrosis in Schistosoma japonicum infection.

We describe the parameters useful in evaluating the development of hepatic fibrosis in Schistosoma japonicum infection, as well as its improvement after treatment with praziquantel (PZQ). Various serologic parameters and ultrasonographic images were examined, and their changes were monitored using rabbits infected with 200 or 300 cercariae of S. japonicum. Infected rabbits were administered one oral treatment of PZQ at a dosage of 100 mg/kg at 6, 12, or 24 weeks after infection. Histopathologic examinations revealed that PZQ had a strong and rapid effect, even on damage that developed long after the infection. The improvement of moderate hepatic fibrosis that developed over 24 weeks after infection was also detected by histopathologic examinations. The serum level of total bile acid was the most sensitive parameter in evaluating the severity of hepatic fibrosis and its improvement after treatment with PZQ. The level of serum procollagen-III-peptide was also useful in evaluating the development of hepatic fibrosis, but not in its improvement. Ultrasonography revealed specific echogenic bands and nodules according to the progress of granuloma formation and fibrosis, and the reversal of these changes could also be observed after treatment with PZQ.

Essential fatty acid synthesis and its regulation in mammals.

The tissue content of highly unsaturated fatty acids (HUFA) such as arachidonic acid and docosahexaenoic acid is maintained in a narrow range by feedback regulation of synthesis. Delta-6 desaturase (D6D) catalyzes the first and rate-limiting step of the HUFA synthesis. Recent identification of a human case of D6D deficiency underscores the importance of this pathway. Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that activates transcription of genes involved with fatty acid synthesis. We recently identified sterol regulatory element (SRE) that is required for activation of the human D6D gene by SREBP-1c. Moreover, the same SRE also mediates the suppression of the D6D gene by HUFA. The identification of SREBP-1c as a key regulator of D6D suggests that the major physiological function of SREBP-1c in liver may be the regulation of phospholipid synthesis rather than triglyceride synthesis. Peroxisome proliferators (PP) induce fatty acid oxidation enzymes and desaturases in rodent liver. However, the induction of desaturases by PP is slower than the induction of oxidation enzymes. This delayed induction may be a compensatory reaction to the increased demand of HUFA caused by increased HUFA oxidation and peroxisome proliferation in PP administration. Recent studies have demonstrated a critical role of peroxisomal beta-oxidation in DHA synthesis, and identified acyl CoA oxidase and D-bifunctional protein as the key enzymes.

Development of the human pontine nuclei: a morphometric study.

The morphometric development of the pontine nuclei in the human fetus from 16 to 40 gestational weeks, in a 2-month-old infant and in a 63-year-old adult were examined employing a serial celloidin section method and computer assisted electronic planimeter. The results of our study can be summarized as follows: (1) the development of the human pontine nuclei accelerated in volume after 32 gestational weeks and continued after birth, (2) neuron numbers remained relatively constant after 27 gestational weeks. It was difficult to clearly distinguish neurons from glia before 27 gestational weeks. The total estimated neuronal numbers were not indicative of the gestational stages in infants 27 gestational weeks and older, (3) individual neurons appeared to continue to develop after 32 gestational weeks in accordance with size, distribution and circularity ratio, (4) many islet-shaped groups of large neurons appeared and were scattered throughout the pontine nuclei after 32 gestational weeks.