Suppurative intracranial complications of sinusitis in adolescence. Single institute experience and review of literature.

The objective of this study was to investigate the outcome of neurosurgical treated children with suppurative intracranial complications (SIC) of sinusitis over the past 28 years in our hospital. We reviewed the cases notes of a series of 11 consecutive paediatric patients, who were subjected to surgery for sinusitis-induced SIC, retrospectively. Eleven children (10 males and only one female) were underwent neurosurgical procedure in our hospital between 1978 and 2006. Their age at the time of diagnosis ranging from 13 to 17 years (mean 15.27 years, SD 1.737). The commonest presenting symptoms were headaches (81.8%) followed by vomiting (45.5%) and swelling of the forehead (45.5%). The most often involved sinus was the frontal sinus (63.6%) and sinus surgery was performed in eight (72.72%) of 11 cases. The neurosurgical procedures carried out included burr hole drainage or aspiration of abscess in five cases, craniotomy and evacuation of empyema in seven cases and craniectomy in two cases. Four (36.4%) of 11 patients had more than one neurosurgical operation due to re-accumulation of pus and worsening of their clinical status. Most common pathogen was Streptococcus species (81.9%), and anaerobes were isolated in three (27.3%) cases. Postoperative antibiotic treatment lasted from 26 to 70 days (mean 45.45 days, SD 15.280). Epilepsy was diagnosed in two patients, postoperatively. During the follow-up period, persistent focal neurological deficits were present in five (45.5%) of 11 patients. Interestingly, five (45.45%) cases occurred over the last 2 years (2005-2006) and the other six over the previous 16 years (1978-2006). Prompt and aggressive medical and neurosurgical intervention is required, aiming to minimize the morbidity and mortality and also to maximize the favourable outcome of those children.

Synthesis and biological evaluation of some new 3 ,4-dihydropyrimidin-4-ones.

Condensation of 5-cyano-2-hydrazino-3-N-methyl-6-phenyl/p-chlorophenyl-3,4-dihydropyrimidin-4-one (3a and 3b) with 2,4-bisalkyl/arylamino-6-chloro-s-triazine (4) gave the corresponding 2,4-bisalkyl/arylamino-6-[5'-cyano-3'-N-methyl]-6'-phenyl/pchlorophenyl-3',4'-dihydropyrimidin-4'-one-2'-yl-hydrazino-s-triazines (5a-n and 6a-n). The compounds 4 have been prepared by the condensation of cyanuric chloride and different alkyl/aryl amines. The reaction between 5-cyano-3-N-methyl-2-methylthio-6-phenyl/p-chlorophenyl-3,4-dihydropyrimidin-4-one (2a and 2b) with hydrazine hydrate furnished 3a and 3b, respectively. The condensation of 6-phenyl/p-chlorophenyl/5-cyano-2-mercapto-3,4-dihydropyrimidin-4-one (1a and 1b) with methyl iodide yielded 2a and 2b, respectively. All the products have been evaluated in vitro for their antimicrobial activity against several microbes and antitubercular activity against Mycobacterium tuberculosis H37 Rv.

Changes in the lipophilicity of the surfaces of Meloidogyne incognita and Haemonchus contortus during exposure to host signals.

The surfaces of plant and animal parasitic nematodes share certain lipids, which seem to be important in the infection process. The surfaces of 2 parasitic nematodes, Meloidogyne incognita and Haemonchus contortus, were activated by different pH buffers to allow the insertion of different fluorescent probes. The lipid analogue PKH26 and the surface charge indicator, cationized ferritin, were used as probes with these nematodes but labelled only the retaining 2nd-stage moulted cuticle of H. contortus 3rd-stage larvae (L3). Shedding of the second moult of H. contortus L3 was also visualized with PKH26 and cationized ferritin. The fluorescent anionic lipid probe 5-N-(octadecanoyl)-aminofluorescein (AF18) was inserted into the epicuticle layer of M. incognita 2nd-stage juveniles (J2) and H. contortus L3, and also of the second moult of H. contortus L3. Incubation with tomato root diffusate caused modifications of the M. incognita surface allowing the insertion of AF18. Fluorescence with AF18 was significantly decreased after treating M. incognita J2 with amiloride, a potent blocker of hydrogen and sodium (H+/Na+) antiporter. No surface fluidity was observed in M. incognita J2 and H. contortus L3 pre-treated with alkaline buffer when the lipid analogue AF18 was used in fluorescence recovery after photobleaching experiments. The significance of these findings to host infection processes is discussed.

Secretion of IL-12 by murine macrophages activated by immunoglobulin receptor-mediated internalization of the surface coat of Trichinella spiralis larvae.

Trichinella spiralis larvae incubated with a rabbit antiserum raised against the larval surface coat bound murine macrophages to the parasite surface. Cell binding was not observed without the antisurface coat serum, or with incubation of larvae in normal rabbit serum, or with antibodies to keyhole limpet haemocyanin which identify a cryptic T. spiralis larval antigen. Cell adherence to the larval surface was lost by treatment of the cells with the lysosomotropic drug primaquine, implicating a receptor-mediated mechanism. Cells adhering to the parasite surface internalized parasite surface coat material, which was subsequently concentrated into endosomes. Culture supernatants from these cells contained enhanced levels of IL-12. Thus, the initial Th1 response to T. spiralis infection may be explained by these data.

The surface coat of infective larvae of Trichinella spiralis.

The surface coat of the infective larvae of the parasitic nematode Trichinella spiralis was characterized with respect to its biophysical properties, morphology and composition. Labelling of larvae with the fluorescent surface probe PKH26 was lost after activation (by incubation in mammalian medium containing trypsin and bile), or following pronase treatment. Electron microscopical examination revealed that pronase treatment resulted in the loss of an amorphous surface layer only, further demonstrating the specificity of PKH26 for the larval surface coat. Surface coat shedding was inhibited by sodium azide and carbonyl cyanide, or by incubation of larvae at 4 degrees C, suggesting the shedding process required metabolic energy. Pre-labelled, unactivated larvae demonstrated continuous slow surface coat shedding and could be re-labelled with PKH26, indicating that the shed coat is replaced in these parasites. However, pre-labelled larvae which were activated failed to re-label with the probe, suggesting that activation provides an irreversible trigger for surface changes. PKH26, therefore, is a useful marker for larval activation. Examination of the shed coat material by scanning electron microscopy revealed 2 types of morphologies; one comprising thin multilaminate sheets and the other of amorphous material with ridges producing a fingerprint-like motif. Western- and lectin-blotting of the shed coat material demonstrated 2 prominent entities; a 90 kDa glycoprotein, which bound Datura stramonium agglutinin and was resistant to N- and O-glycanase treatment and a 47-60 kDa set of protein(s). Analysis of the surface lipids by electrospray mass spectometry revealed the presence of lysophosphatidic acid (lysoPA, C14:2) and an unidentifiable component of 339.4 Da. These two lipids constituted 36.9% and 36% by mass of surface coat lipids respectively. The presence of lysoPA was confirmed by thin layer chromatography, which also detected phosphatidic acid (PA). The polar lipids detected in solvent rinses of intact parasites by electrospray mass spectrometry were PI (C48:4), PE (C40:4 and C38:4), PS (C40:4), lysoPC (C20:2 and C18:2) and lysoPA (C14:2). These observations are discussed with respect to the role of the surface coat and its shedding in the T. spiralis host-parasite relationship.

Schistosomes: unanswered questions on the basic biology of the host-parasite relationship.

As is the case in many parasite infections, research into schistosomiasis has not yet yielded a vaccine and, although chemotherapy with praziquantel is very effective, the mechanism of action of this drug is unknown. John Kusel and colleagues here suggest that an understanding of basic biological phenomena, such as the role of Ca(2+) in skin penetration and the function of the adult excretory system, might lead to important breakthroughs. Other crucial questions are also addressed, with the hope of stimulating debate. They invite suggestions and correspondence from others working in related fields.

Probing the nematode surface.

The surface of parasitic nematodes has been well studied with respect to its structural and immunological properties, but little is known about its biophysical nature and the role this plays in the host-parasite relationship. In this article, Clare Roberts and Jay Modha highlight some biophysical features of nematode surfaces and discuss their recent findings regarding mechanisms controlling surface-associated biophysical phenomena observed in parasitic nematodes during infection or culture in medium simulating the mammalian host environment. The nematode surface is distinct from the plasma membrane, nevertheless some parallel features exist and are described.

Induction of surface fluidity in Trichinella spiralis larvae during penetration of the host intestine: simulation by cyclic AMP in vitro.

The lateral diffusion (DL) properties of the fluorescent lipid probe 5-N (octadecanoyl) aminofluorescein (AF18) inserted into the surface of muscle-stage larvae of Trichinella spiralis were investigated by fluorescence recovery after photo-bleaching. AF18 was not free to diffuse laterally in dormant larvae, and this remained unchanged after larval activation in vitro with trypsin and bile. However, a significant increase in surface fluidity of the probe was demonstrated (%R = 74.5; DL = 11.5 x 10(-9) cm2/sec) when larvae invaded intestinal epithelial tissue following oral infection of mice. Membrane-permeant photoactivatable caged cyclic AMP was used to analyse the putative mechanism responsible for this increase in lateral diffusion in the parasite surface. Although incubation of larvae with 1-50 microM caged cAMP had no effect on surface fluidity, incubation with 100 microM caged cAMP induced a substantial increase in the lateral mobility of AF18 (%R = 64.3; DL = 8.3 x 10(-11) cm2/sec) immediately following photo-activation of the caged messenger. This induced fluidity, however, was transient and the larval surface reverted to immobility within 15 min. These observations constitute the first reported measurement of the fluid properties of the surface of intracellular parasites, the first demonstration of the parasite surface fluidity altering as a result of host cell invasion and the first indication of a mechanism underlying changes in surface fluidity in parasitic helminths.

Schistosomes and serpins: a complex business.

The view of the schistosome host-parasitic relationship has changed in the past two decades. Previously, it was thought the parasite simply defended itself in the face of a hostile host environment. However, it is now realized that the host-parasite interaction is much more of a dynamic interplay, where the parasite is able to exploit host homeostatic mechanisms for survival, maturity and transmission. Here, Jay Modha, Clare Roberts and John Kusel discuss the recent identification of serine protease inhibitors (serpins) on the schistosome surface and suggest how their properties might be exploited by the parasite.

Praziquantel: an urgent and exciting challenge.

The anthelmintic drug praziquantel has proved useful in the treatment of schistosomiasis. The precise mechanism by which praziquantel kills the parasites has yet to be elucidated. Here, John Kusel and colleagues review the current theories on praziquantel action and suggest future avenues for research, which becomes urgent in the light of some reports of drug resistance.

A role for second messengers in the control of activation-associated modification of the surface of Trichinella spiralis infective larvae.

The involvement of second messengers in the control of activation-induced changes to the surface of Trichinella spiralis infective larvae was investigated using membrane-permeant photo-activatable 'caged' compounds to alter intracellular levels of inositol trisphosphate (IP3), calcium ions (Ca2+) and cyclic AMP (cAMP). Activation of larvae by incubation in culture medium containing trypsin and bile was followed by the loss of the surface coat labelled with the fluorescent PKH26 lipid probe and this correlated with the reciprocal acquisition of surface lipophilicity detected using the fluorescent lipid probe octadecanoyl aminofluorescein (AF18). Optimal surface coat shedding and AF18 insertion was also achieved following photolysis of caged mediators liberating IP3, Ca2+ or cAMP within the parasite. Chelation of Ca2+, however, abolished the effects of larval activation. Nevertheless, addition of cAMP (but not IP3) to Ca(2+)-depleted larvae overcame this inhibition and restored AF18 insertion to levels achieved by activated parasites. Therefore, the existence of a linear second messenger pathway involving the sequential release of IP3, Ca2+ and then cAMP is likely.

Schistosoma mansoni host-parasite relationship: interaction of contrapsin with adult worms.

Contrapsin, a serine protease inhibitor (serpin) present in mouse serum, was compared with that found in adult Schistosoma mansoni worm homogenates, which although immunologically identical to contrapsin in mouse serum, had a higher molecular weight in Western blotting. Immunolocalization studies demonstrated parasite-associated contrapsin on the surface and interstitial cells of adult male worms. After extraction of these parasites with Triton X-114, contrapsin was found in the aqueous phase of the detergent, suggesting it is unlikely to be an integral membrane protein. Treatment of adult worms with deoxycholate resulted in a change in the electrophoretic behaviour of worm-derived contrapsin. Parallel studies with trypsin suggested this was due to interaction of the serpin with a protease. Using porcine pancreatic trypsin as a model for a putative schistosome protease reacting with contrapsin, we have shown that trypsin, following complex formation with contrapsin, loses immunogenicity. Thus, when contrapsin-trypsin complexes were used as immunogen, the resulting antisera contained antibodies to contrapsin and contrapsin-trypsin complexes only, and none to native trypsin. Thus, epitopes characterizing native trypsin were presumably either masked following complex formation with contrapsin, or their processing and presentation to antigen presenting cells was suppressed, so that an antibody response was not mounted against them. These observations encourage speculation that S. mansoni may be elaborating an immune evasion strategy whereby immunologically sensitive proteases are first complexed with host serpins, which would render them immunogenically inert, and then cleared from the circulation by the host's reticulo-endothelial system. In this way the immune system would be unable to 'see' sensitive parasite proteases sufficiently to mount a response against the parasite.

Characterization of a major surface-associated excretory-secretory antigen of Trichinella spiralis larvae with antibodies to keyhole limpet haemocyanin.

A multi-subunit antigen (native M(r) > 200 kDa, reduced M(r) 97-100 kDa) has been identified in homogenates of Trichinella spiralis larvae using affinity-purified rabbit anti-keyhole limpet haemocyanin (KLH) antibodies and its cross-reactivity with KLH was confirmed by competition blotting. The antigen was not present at the larval surface but was exposed after treatment of the larvae with the detergent cetyltrimethyl ammonium bromide (CTAB) which removed the surface coat. This correlated with a significant decrease in insertion of the surface-restricted fluorescent lipid probe AF18, indicating that the surface coat must be lipidic in nature. Unlike KLH, the larval antigen blotted onto nitrocellulose was itself periodate insensitive. Periodate treatment of whole larvae, however, resulted in shedding of the surface, to which anti-KLH antibodies then bound intensely. Anti-KLH antibodies also recognized three (49, 55, 108 kDa) of the four most dominant antigens in excretory-secretory (ES) products of cultured larvae, whose excretion-secretion was increased with CTAB. The nature, location and function of the antigen is discussed.

Praziquantel-induced exposure of Schistosoma mansoni alkaline phosphatase: drug-antibody synergy which acts preferentially against female worms.

The efficacy of praziquantel-treatment of murine Schistosoma mansoni-infections can be enhanced by concurrent administration of rabbit anti-sera with specificity for parasite antigens. Monospecific rabbit serum raised against S. mansoni worm alkaline phosphatase, that was reactive with the enzyme on the drug-treated female surface, was found to significantly and preferentially increase the mortality of female worms by PZQ. Immunoglobulins purified from the anti-alkaline phosphatase antiserum inhibited 54% of schistosome alkaline phosphatase enzymatic activity on the surface of praziquantel-treated worms. We propose that synergistic antibody-mediated death of drug-damaged worms is a consequence of the inhibition of drug-exposed alkaline phosphatase on the female worm surface by passively transferred antibody.

Complex formation of human alpha-1-antitrypsin with components in Schistosoma mansoni cercariae.

Human alpha-1-antitrypsin (alpha 1-AT) was incubated with an extract of Schistosoma mansoni cercariae or porcine pancreatic elastase and analysed by immunoelectrophoresis and Western blotting. The inhibitor was shown to form complexes with components in S. mansoni cercariae in the same way as elastase. The role of alpha 1-AT in S. mansoni infection is discussed.

Controlled high-level expression of the lon gene of Escherichia coli allows overproduction of Lon protease.

Lon protease from Escherichia coli is an ATP-dependent protease which plays important roles in regulating the levels of specific proteins and in eliminating abnormal proteins. A major problem of working with Lon protease, the inability to substantially overproduce the enzyme, has been overcome by placing the lon gene under the control of an inducible trp promoter within a copy-number-controllable plasmid. Induction resulted in higher levels of production of the protease (approximately 100 micrograms/ml of cell culture) than were previously possible. The enzyme has been purified to apparent homogeneity and shown to possess the characteristic ATP-dependent proteolytic activity. Sequence verification during DNA manipulations revealed differences from two previously published sequences for the lon gene.

The immune dependence of chemotherapy.

In this review, Mike Doenhoff and colleagues discuss the immune dependency of chemotherapy and the consequences for drug resistance. They also consider the implications for the control of infections that are relatively unresponsive to drugs, such as opportunistic infections in immunosuppressed patients.

Proteases in the schistosome life cycle: a paradigm for tumour metastasis.

Cancers and parasites have a number of properties in common, particularly those that relate to their respective capacities to evade host defence mechanisms. This review highlights the similarities between metastatic tumours and schistosomes in particular, and describes the role that proteases may have in the migration, growth, survival and transmission of the different stages of the schistosome life-cycle in the vertebrate host. An elastase-like serine protease of schistosome larvae has been particularly well characterized, and its substrate profile and other properties are indicative of a role in facilitating migration of the parasite through skin tissue early after infection. The primary structures of a cathepsin B-like enzyme, and a putative 'haemoglobinase' found in adult worms have also recently been derived, these enzymes being responsible for degradation of haemoglobin in erythrocytes upon which the adults feed. Adult schistosome worms reside and produce eggs intravascularly, and the processes that mediate the extravasation and subsequent migration of the egg through host tissue are dependent on both blood platelets and the immune response. Fibrino(geno)lytic enzymatic activity that is present in the egg could modulate the thrombogenic potential that eggs might have as a result of their capacity to cause platelet aggregation in vitro and in vivo. The roles of other proteases and peptidases that have been found in schistosome larvae, worms and eggs are less clear. Some of these enzymes may modulate immunological and haemostatic defence mechanisms and thus prolong survival of the parasite, and the consequences of the interactions between schistosomes and host protease inhibitors could also be immune modulatory.