Conservative management of neurocysticercosis in a patient with hematopoietic stem cell transplantation: a case report and review.

Neurocysticercosis, an infection of the central nervous system with the larval stage of the cestode Taenia solium, is common in developing countries but its occurrence and management in allogeneic hematopoietic stem cell transplantation (HSCT) has not been reported previously, to our knowledge. We report the case of an immigrant female patient who underwent a matched-related allogeneic HSCT for acute lymphoblastic leukemia and was incidentally found to have a solitary viable neurocysticercosis lesion. However, despite severe immunosuppression, the size of the cyst did not increase. More importantly, restoration of the immune system did not induce significant inflammation or seizures. Subsequent follow-up demonstrated complete resolution of the neurocysticercosis lesion. Thus, in the setting of HSCT, an asymptomatic patient with a single neurocysticercosis lesion was successfully managed without the use of anthelmintics, steroids, or anti-epileptics.

Sensitive in vitro system to assess morphological and biochemical effects of praziquantel and albendazole on Taenia solium cysts.

Neurocysticercosis resulting from Taenia solium infections is a major cause of adult-acquired seizures worldwide. Disease is caused by larval cysts, and treatment consists of the anthelmintic drugs albendazole or praziquantel. There are no standard methods to assess drug activity to T. solium cysts in vitro. Morphological, functional, and biochemical changes that might reflect damaging (inhibiting, cytotoxic) drug effects were analyzed after exposure of cysts to albendazole sulfoxide (ABZ-SO), the major active metabolite of the drug in vivo, praziquantel (PZQ), or combinations of both. PZQ exposure led to a decrease in cyst size and inhibition of evagination, whereas ABZ-SO exposure resulted in minimal changes. Alkaline phosphatase (AP) is normally secreted by cysts, and both drugs inhibited AP secretion at concentrations of 5 and 50 ng/ml for PZQ and ABZ-SO, respectively. Some combinations of both drugs resulted in additive and/or synergistic activities. Parasite-specific antigen, detected in the cerebrospinal fluid and blood of infected patients, is also normally secreted by T. solium cysts. Antigen secretion was similarly inhibited by ABZ-SO and PZQ and a combination of both drugs, suggesting that inhibition of secretion is a common downstream consequence of the activities of both drugs. These studies establish quantitative methods to measure in vitro anthelmintic activity and suggest combination therapy with ABZ-SO and PZQ may have clinical benefit.

Functional binding between Gbeta and the LIM domain of Ste5 is required to activate the MEKK Ste11.

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, the pheromones that induce haploid cells of opposite cell types to mate activate the Gbeta and Ggamma subunits of a heterotrimeric G protein. These subunits signal through the PAK kinase Ste20 to activate a mitogen-activated protein (MAP) kinase cascade comprising the MEKK Ste11, the MEK Ste7 and two MAP kinases, Fus3 and Kss1. The pathway requires Ste5, a scaffold protein that tethers the MAP kinase cascade enzymes into a high molecular weight complex. Ste5 is thought to associate with Gbeta in a pheromone-independent manner, but it is not known if this interaction affects signaling. RESULTS: A ste5C180A mutant - which expresses Ste5 disrupted in the LIM domain, a putative metal-binding motif that has been proposed to be essential for Ste5 oligomerization - could not transmit the pheromone signal from Gbeta through Ste20 to Ste11. The Ste5C180A protein was impaired in binding Gbeta, although it could oligomerize, bind Ste11, Ste7 and Fus3, facilitate the basal activation of Ste11, and relay the Ste11 signal to MAP kinases. Ste5 bound to Gbeta in a pheromone-dependent manner and preferentially associated with a phosphorylated form of Gbeta in wild-type and ste20Delta, but not in ste5C180A, strains. CONCLUSIONS: Pheromone induces binding of Gbeta to Ste5 through its LIM domain. This binding is essential for activation of Ste11 and is distinct from the ability of Ste5 to oligomerize or to serve as a scaffold and relay the signal from Ste11 to the MAP kinases. Pheromone also induces Ste5-dependent phosphorylation of Gbeta.

Iron chelation as a possible mechanism for aspirin-induced malondialdehyde production by mouse liver microsomes and mitochondria.

To investigate the possibility that lipid peroxidation is the mechanism responsible for aspirin-induced liver damage, pure neutralized acetylsalicylic acid (ASA), 0.6-90.9 mM, was added to calcium-aggregated mouse liver microsomes followed by incubation in NADPH buffer at 37 degrees C for 60 min and subsequent measurement of malondialdehyde (MDA). MDA production at ASA concentrations from 1.2 to 4.6 mM was greater than control (P less than 0.004). Peak MDA values were observed with 4.6 mM ASA, 39.58 +/- 6.73 nmol MDA/mg protein vs. 16.16 +/- 2.85 (P less than 0.004). Higher concentrations of ASA were inhibitory compared with the value at 4.6 mM (P less than 0.001). Aspirin had similar effects on MDA production by mouse liver mitochondria. MDA production with either ASA or buffer was completely suppressed by the potent iron-chelating agents desferrioxamine and alpha,alpha' dipyridyl when these were added to the microsomal preparations. Since MDA production in this system is known to be affected by iron-chelating agents (enhanced at low concentration, inhibited at higher concentration), the iron-chelating properties of ASA were investigated. Conductivity titration curves of Fe(OH)3 added to water or ASA suggested that the ASA was complexing with iron. The presence of an iron-ASA complex was established by high pressure liquid chromatographic analysis of the solution from this study. We conclude that aspirin enhances MDA production by hepatic microsomes and mitochondria via an aspirin-iron chelate and that this represents at least one mechanism by which aspirin may produce liver damage.

Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset.

The immunological mechanisms involved in maintenance of an asymptomatic microfilaremic state (MF) in patients with lymphatic filariasis remain undefined. MF patients have impaired filarial antigen (Ag)-specific lymphocyte proliferation and decreased frequencies (Fo) of Ag-specific T cells, and yet elevated serum IgE and antifilarial IgG4. To investigate the mechanism of Ag-specific anergy in MF patients in contrast to amicrofilaremic individuals with chronic lymphatic obstruction (CP), the Fo of Ag-specific lymphocytes from peripheral blood mononuclear cells secreting either IL-4 or IFN-gamma were assessed by filter spot enzyme-linked immunosorbent assay, and IL-10 and transforming growth factor-beta (TGF-beta) mRNA transcript levels were assessed by a semiquantitative reverse transcriptase polymerase chain reaction technique. The Fo of filaria-specific IL-4-secreting lymphocytes were equivalent in both MF (geometric mean [GM] = 1:11,700) and CP (GM = 1:29,300 P = 0.08), whereas the Fo of IFN-gamma-secreting lymphocytes were lower in MF (GM = 1:39,300) than in CP (GM = 1:4,200, P < 0.01). When the ratio of IL-4/IFN-gamma (T helper type 2 [Th2]/Th1)-secreting cells was examined, MF subjects showed a predominant Th2 response (8:1) compared with a Th1 response in CP individuals (1:4). mRNA transcript levels of IL-10 were also significantly elevated in MF compared with CP individuals (P < 0.01). Further, IL-10 and TGF-beta were shown to have a role in modulating the Ag-specific anergy among MF subjects, in that neutralizing anti-IL-10 or anti-TGF-beta significantly enhanced lymphocyte proliferation response (by 220-1,300%) to filarial Ags in MF individuals. These findings demonstrate that MF subjects respond to parasite antigen by producing a set of suppressive cytokines that may facilitate persistence of the parasite within humans while producing little clinical disease.

The SH3-domain protein Bem1 coordinates mitogen-activated protein kinase cascade activation with cell cycle control in Saccharomyces cerevisiae.

The mating mitogen-activated protein kinase (MAPK) cascade has three major outputs prior to fusion: transcriptional activation of many genes, cell cycle arrest in the G1 phase, and polarized growth. Bem1 localizes near the cortical actin cytoskeleton and is essential for polarized growth during mating. Here we show that Bem1 is required for efficient signal transduction and coordinates MAPK cascade activation with G1 arrest and mating. bem1delta null mutants are defective in G1 arrest and transcriptional activation in response to mating pheromone. Bem1 protein stimulates Fus3 (MAPK) activity and associates with Ste5, the tethering protein essential for activation of the MAPK kinase kinase Ste11. Bem1-Ste5 complexes also contain Ste11, Ste7 (MAPK kinase), and Fus3, suggesting that Ste5 localizes the MAPK cascade to Bem1. Strikingly, Bem1 also copurifies with Far1, a Fus3 substrate required for G1 arrest and proper polarized growth during mating. These and other results suggest that Bem1 may cross-link the Ste5-MAPK cascade complex to upstream activators and specific downstream substrates at the shmoo tip, thus enabling efficient circuitry for G1 arrest and mating.

Characterization of Fus3 localization: active Fus3 localizes in complexes of varying size and specific activity.

The MAP kinase Fus3 regulates many different signal transduction outputs that govern the ability of Saccharomyces cerevisiae haploid cells to mate. Here we characterize Fus3 localization and association with other proteins. By indirect immunofluorescence, Fus3 localizes in punctate spots throughout the cytoplasm and nucleus, with slightly enhanced nuclear localization after pheromone stimulation. This broad distribution is consistent with the critical role Fus3 plays in mating and contrasts that of Kss1, which concentrates in the nucleus and is not required for mating. The majority of Fus3 is soluble and not bound to any one protein; however, a fraction is stably bound to two proteins of approximately 60 and approximately 70 kDa. Based on fractionation and gradient density centrifugation properties, Fus3 exists in a number of complexes, with its activity critically dependent upon association with other proteins. In the presence of alpha factor, nearly all of the active Fus3 localizes in complexes of varying size and specific activity, whereas monomeric Fus3 has little activity. Fus3 has highest specific activity within a 350- to 500-kDa complex previously shown to contain Ste5, Ste11, and Ste7. Ste5 is required for Fus3 to exist in this complex. Upon alpha factor withdrawal, a pool of Fus3 retains activity for more than one cell cycle. Collectively, these results support Ste5's role as a tether and suggest that association of Fus3 in complexes in the presence of pheromone may prevent inactivation in addition to enhancing activation.

(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets.

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .

Functional expression of the cystic fibrosis transmembrane conductance regulator in yeast.

Recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) has been produced in a Saccharomyces cerevisiae expression system used previously to produce transport ATPases with high yields. The arrangement of the bases in the region immediately upstream from the ATG start codon of the CFTR is extremely important for high expression levels. The maximal CFTR expression level is about 5-10% of that in Sf9 insect cells as judged by comparison of immunoblots. Upon sucrose gradient centrifugation, the majority of the CFTR is found in a light vesicle fraction separated from the yeast plasma membrane in a heavier fraction. It thus appears that most of expressed CFTR is not directed to the plasma membrane in this system. CFTR expressed in yeast has the same mobility (ca. 140 kDa) as recombinant CFTR produced in Sf9 cells in a high resolution SDS-PAGE gel before and after N-glycosidase F treatment, suggesting that it is not glycosylated. The channel function of the expressed CFTR was measured by an isotope flux assay in isolated yeast membrane vesicles and single channel recording following reconstitution into planar lipid bilayers. In the isotope flux assay, protein kinase A (PKA) increased the rate of 125I- uptake by about 30% in membrane vesicles containing the CFTR, but not in control membranes. The single channel recordings showed that a PKA-activated small conductance anion channel (8 pS) with a linear I-V relationship was present in the CFTR membranes, but not in control membranes. These results show that the human CFTR has been expressed in functional form in yeast. With the reasonably high yield and the ability to grow massive quantities of yeast at low cost, this CFTR expression system may provide a valuable new source of starting material for purification of large quantities of the CFTR for biochemical studies.

Wheat germ supplement reduces cyst and trophozoite passage in people with giardiasis.

The protozoan parasite Giardia lamblia is a major cause of waterborne enteric disease worldwide. Lectins are proteins that bind to carbohydrate (sugar) moieties. Potential targets for lectins are found on the surface of most single-celled organisms. Modest concentrations of wheat germ agglutinin (WGA) have been shown to inhibit G. lamblia excystation and trophozoite growth in vitro and can reduce cyst passage in mice infected with the closely related protozoan parasite, G. muris. Commercial preparations of wheat germ (WG) contain 13-53 microg of WGA per gram. We performed a double-masked, placebo-controlled study of dietary supplementation with WG in 63 subjects with giardiasis in Montreal and Lima (25 asymptomatic patients passing cysts; 38 patients with symptoms). Asymptomatic subjects received WG (2 g, 3 times a day) or placebo (cornstarch, 2 g, 3 times a day) for 10 days, followed by metronidazole (250 mg 3 times a day) for 7 days. Symptomatic subjects received metronidazole (250 mg 3 times a day) plus either WG or placebo for 7 days. Stool specimens were collected every day (Montreal) or every other day (Lima) for 10 days and on Day 35 for microscopic examination and coproantigen determination. Subjects kept a diary of symptoms for 10 days after recruitment. In asymptomatic subjects, both cyst passage and coproantigen levels were reduced by approximately 50% in those taking WG compared with the placebo group (P < 0.01 and P = 0.06, respectively). In symptomatic subjects, cyst passage and coproantigen levels fell precipitously in response to metronidazole therapy, and there were no clinically important differences between those receiving supplemental WG or placebo. However, symptoms appear to have resolved more rapidly in the subjects taking WG in addition to metronidazole. The WG supplement was well tolerated in both symptomatic and asymptomatic subjects. These data suggest that components of WG, possibly WGA, either alone or in combination with antiprotozoal agents, can influence the course of human giardiasis.

Vanadate-resistant mutants of Candida albicans show alterations in phosphate uptake.

Phosphate uptake studies in different strains of the dimorphic pathogenic yeast Candida albicans were undertaken to show that this yeast actively transported phosphate with an apparent Km in the range of 90-170 microM. The uptake was pH dependent and derepressible under phosphate starvation. Vanadate-resistant (van) mutants of C. albicans showed a 20-70% reduction in the rate of phosphate uptake in high phosphate medium and was associated with an increased Km and reduced Vmax. The magnitude of derepression under phosphate starvation was different between van mutants. These results demonstrate that van mutants may have developed resistance by modifying the rate of entry of vanadate.

UVB induces IL-12 transcription in human keratinocytes in vivo and in vitro.

Human epidermal cells produce a wide range of cytokines, including those characteristic of Th2-like responses such as interleukin (IL)-4 and IL-10. As well, keratinocytes have recently been shown to produce Th1-like cytokines such as IL-12. Exposure to UVB has profound effects on the skin and systemic immune system, which is in part mediated by secretion of tumor necrosis factor (TNF)-alpha by epidermal cells. Because IL-12 induces production of TNF-alpha by certain cells of the immune system, we sought to determine whether UVB is an inducer of IL-12 gene expression in epidermal cells. Human epidermal cells were exposed to UVB radiation in vivo, isolated by suction blister technique and trypsinization and transcription of the IL-12 p35 and p40 chains was examined by RT-PCR. We found the p35 chain of IL-12 to be constitutively expressed and the p40 chain inducible by UVB irradiation. Because epidermis consists of a heterogenous cell population with distinct cytokine repertoires, we sought to determine the cellular source of the IL-12 message after UVB exposure. After depleting UVB-exposed epidermal cells for DR+ cells, no reduction in the IL-12 activity was detected, suggesting that keratinocytes are a source of IL-12 transcripts in UVB-exposed human epidermis. This was supported by the up-regulation of IL-12 p40 transcripts in UV-irradiated cultured keratinocytes that were devoid of DR+ cells. Up-regulation of IL-12 p40 gene expression by UVB as demonstrated here, taken together with the finding that keratinocytes also up-regulate IL-10 transcription, suggests that there is a complex interplay between Th1- and Th2-like epidermis-derived cytokines following exposure to UVB.

Gas chromatographic determination of pollutants in the chlorination and caustic extraction stage effluent from the bleaching of a bamboo pulp.

The gas chromatographic detection and quantitative determination of various chlorophenolics as well as resin and fatty acids have been carried out in the chlorination and caustic extraction stage effluents generated in the laboratory by bleaching a bamboo pulp. A number of chlorinated phenols, catechols, guaiacols, syringaldehydes and resin acids as well as non-chlorinated saturated and unsaturated fatty acids together with resin acids have been detected. The concentration of various compounds detected have also been compared with the reported (96)LC(50) values.

Febrile illness caused by parasites.

Febrile patients in whom a parasitic etiology is suspected can be classified broadly into two categories for the purpose of diagnostic evaluation based on the presence or absence of accompanying eosinophilia. Generally, the absence of eosinophilia indicates a protozoan etiology while the presence of eosinophilia is associated with tissue-invasive helminthic infections. The history and physical examination often points to a specific diagnosis that can be confirmed by appropriate laboratory tests.

Immunoregulation in human lymphatic filariasis: the role of interleukin 10.

In humans with lymphatic filariasis microfilaremia is associated with a parasite antigen-specific hyporesponsiveness when assessed by cell proliferation and secretion of interleukin-2 and interferon-gamma. Hyporesponsiveness in these individuals is not only parasite antigen-specific but appears to be limited to Th1-type responses. Th2 mediated responses such as IL-5 secretion and IgE antibody production to parasite antigens are generally strong and usually no different than those seen in immunologically more reactive amicrofilaremic individuals with chronic lymphatic pathology. The mechanisms by which Th1 responses are inhibited have not yet been elucidated, but some studies suggest that down-regulatory cytokines such as IL-10 may be involved in this process. Mononuclear cells from microfilaremic individuals have been found to secrete greater quantities of IL-10 spontaneously and in response to parasite antigens. In this review, mechanisms by which IL-10 may be induced by the parasite and the mode by which IL-10 may regulate parasite antigen-specific Th1 responses in these individuals are discussed.

Site-directed mutagenesis of the cysteine residues in the Neurospora crassa plasma membrane H(+)-ATPase.

A high-yield yeast expression system for site-directed mutagenesis of the Neurospora crassa plasma membrane H(+)-ATPase has recently been reported (Mahanty, S. K., Rao, U. S., Nicholas, R. A., and Scarborough, G. A. (1994) J. Biol. Chem. 269, 17705-17712). Using this system, each of the eight cysteine residues in the ATPase was changed to a serine or an alanine residue, producing strains C148S and C148A, C376S and C376A, C409S and C409A, C472S and C472A, C532S and C532A, C545S and C545A, C840S and C840A, and C869S and C869A, respectively. With the exception of C376S and C532S, all of the mutant ATPases are able to support the growth of yeast cells to different extents, indicating that they are functional. The C376S and C532S enzymes appear to be non-functional. After solubilization of the functional mutant ATPase molecules from isolated membranes with lysolecithin, all behaved similar to the native enzyme when subjected to glycerol density gradient centrifugation, indicating that they fold in a natural manner. The kinetic properties of these mutant enzymes were also similar to the native ATPase with the exception of C409A, which has a substantially higher Km. These results clearly indicate that none of the eight cysteine residues in the H(+)-ATPase molecule are essential for ATPase activity, but that Cys376, Cys409, and Cys532 may be in or near important sites. They also demonstrate that the previously described disulfide bridge between Cys148 and Cys840 or Cys869 plays no obvious role in the structure or function of this membrane transport enzyme.

Thermal response of skin to application of localized pressure.

Pressure was applied to a small circular area of the skin over the trochanter of human subjects for variable time periods. The local skin temperature response was monitored during and after pressure application. Pressures of 100, 200, and 300mmHg were applied for time periods of between 11 and 67 minutes. Upon the release of pressure the local skin temperature rose quickly and then gradually decreased. Peak temperature responses occurred at between 3 and 5 minutes after pressure release. Larger pressure and longer application times produced larger peak temperature increases, with the largest individual increase being 1.9C. The data derived from the study tend to quantify what clinicians have long appreciated in a qualitative sense, especially in areas where decubitus ulcers most often occur.