Mannose-binding Protein Recognizes Glioma Cells: In vitro Analysis of Complement Activation on Glioma Cells via the Lectin Pathway

The lectin pathway is a novel pathway for activation of the complement cascade, which is initiated by the binding of mannose-binding protein (MBP) to its carbohydrate ligands. We investigated whether the complement system was activated in vitro by glioma cells through this pathway to the C3 level. MBP was found to bind to all six glioma cell lines tested by using flow cytometric analysis. Binding of a complex of MBP-associated serine protease and MBP was observed in two of the cell lines examined, thereby resulting in C4 consumption. Activation of C3 was hemolytically evaluated in these two lines. C3 consumption was also observed in one. Based on these results, it is likely that recognition by MBP followed by complement activation occurs in certain glioma cell lines.     

Systemic toxicity in mice induced by localized porphyrin photodynamic therapy

An unexpected high level of acute lethality has been documented following Photofrin II-mediated photodynamic therapy (PDT) treatments which were localized to the hind leg of normal and tumor-bearing mice. Doses of PDT which induced lethality (10 mg/kg Photofrin II, 200-500 J/cm2) were in the range of doses required to obtain murine tumor cures. The percentage of lethality was proportional to the total light dose but was inversely proportional to the dose rate of delivered light. Comparable levels of acute toxicity were observed in four pigmented mouse strains (C57BL/6J, C3H/HeJ, DBA/1, and DBA/2) and in two albino mouse strains (BALB/c and Swiss Webster). Decreased sensitivity to PDT-induced lethality was observed in two pigmented mouse strains (B10D2/OSN and B10D2/NSN). The administration of warfarin, aspirin, indomethacin, or antihistamine had significant protective effects in terms of decreasing PDT-induced lethality. However, injection of cobra venom factor (to deplete C3 and C5 of the complement system) did not alter the lethality mediated by PDT. Histological profiles obtained 24 h following PDT demonstrated vascular congestion in the liver, kidney, lung, and spleen. Significant decreases in removable blood volume, core temperature, and spleen weight were also observed within 24 h of localized PDT treatment. These results indicate that PDT-induced lethality is consistent with a traumatic shock syndrome and suggest that endogenous vasoactive mediators of shock such as prostaglandins, thromboxanes, and histamine are associated with the lethality induced by localized PDT in mice.     

Effect of Zataria multiflora Boiss. essential oil on colony morphology and ultrastructure of Aspergillus flavus

The mode of inhibitory action of Zataria multiflora Boiss. essential oil (EO) on the fungus, Aspergillus flavus, was studied by colony morphology examination, light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The EO at concentrations used in this study suppressed the size of the colony as well as sporulation. SEM of mycelia treated with given concentrations of EO showed morphological alterations ranging from loss of turgidity and uniformity of mycelia at low concentrations of EO to evident destruction of the hyphae at higher concentration of EO. Semi-thin sections of mycelia exposed to different concentrations of EO were analysed by light microscopy and revealed that the major change at level as low as 50 ppm of EO was limited to vacuolisation of cytoplasm resulting in cell swelling, while at higher concentrations, detachment of the cell membrane from the cell wall, deformation of mycelia and shedding the cytoplasm from the cell were the main alterations. These damages were well documented by TEM, which showed that the main sites of action of EO were the plasma membrane and cell wall. In conclusion, morphological and structural changes observed in this study may be one of the mechanisms involved in growth inhibition of the fungi and reducing aflatoxin production.     

Comparative acute nephrotoxicity of Penicillium aurantiogriseum in rats and hamsters.

Air-dried mycelium of Penicillium aurantiogriseum2, grown as a surface culture on yeast extract-sucrose medium, was incorporated into powdered diet and fed to rats and hamsters for different periods up to 28 days. At intervals, animals were anaesthetized and the kidneys fixed in situ by perfusion. In rats, the fungus produced scattered exfoliation of pyknotic cells and an increased frequency of mitotic figures involving the pars recta segment of proximal tubular epithelium. This lesion was detectable as early as three days after beginning of treatment and was well developed by 14 days. No degenerative tubular change or mitogenic effect was observed in hamsters, even after feeding for 35 days; and there was no apparent renal pelvic or interstitial lesion in either species.     

Cyclic Adenosine 3'5' Monophosphate (c-AMP) as a Factor in Phase Morphogenesis of Blastomyces dermatitidis

Summary: The intracellular concentration of cyclic adenosine 3'5' monophosphate (c-AMP) was found to be 1.3 times higher in the mycelium of Blastomyces dermatitidis than that found in its yeastlike growth form. Yeastlike cells of the fungus grown at the preferential temperature of 37°C reverted to mycelial phase growth in the presence of the c-AMP phosphodiesterase inhibitors theophylline or 3-isobutyl-1-methylxanthine. The intracellular activity sites of c-AMP phosphodiesterase in converting yeastlike cells were determined by electron cytochemical means. It is believed that intracellular levels of c-AMP can critically influence phase morphogenesis of B. dermatitidis.
Zusammenfassung: Die intrazelluläre Konzentration von c-AMP im Mycelium von Blastomyces dermatitidis ist 1,3mal höher als in seiner Hefe-ähnlichen Form. Hefe-ähnliche Zellen des Pilzes, gezüchtet beim Temperaturoptimum von 37°C, wechselten in die Mycelphase über, wenn ein Inhibitor der c-AMP-Phosphodiesterase wie Theophyllin oder 3-Isobutyl-1-Methylxanthin zugegen war. Die intrazellulären Aktivitätszentren der c-AMP-Phosphodiesterase in diesem Prozeß wurden mit elektronencytochemischen Methoden bestimmt. Es kann angenommen werden, daß die intrazelulläre Konzentration von c-AMP die Phasenmorphogenese von Blastomyces dermatitidis kritisch beeinflußt.     

Plasma therapy of primary rat mammary carcinoma: dependence of consumption of C3 during absorption of plasma with sepharose derivatives on the anticoagulant

A previous study demonstrated inhibition of growth of primary rat mammary carcinomas after infusion of tumor-bearer plasma absorbed against Sepharose derivatives. In this report we have quantitated changes in individual complement components that occur during absorption of rat plasma with Sepharose derivatives and defined optimal conditions for consumption of the third component of complement (C3) (other complement components defined similarly). The concentration of functionally active C1 to C9 was measured before and after absorption in plasmas from both normal rats and rats with mammary tumors. C3 activity in plasmas from normal and tumor-bearing rats was reduced (consumed) during absorption under appropriate conditions with Sepharose 4B, inactivated CNBr Sepharose, or Protein A-Sepharose. The concentration of functionally active C1 and C4 did not decrease significantly during absorption with Sepharose derivatives. Consumption of C3 in rat plasma was influenced by the anticoagulant and by the time and temperature of incubation with Sepharose derivative. C3 consumption in rat plasma anticoagulated with acid citrate dextrose solution was variable; addition of Mg2+ (5 mM) to plasma anticoagulated with acid citrate dextrose solution augmented C3 consumption. There was no C3 consumption in plasma anticoagulated with ethylenedinitrilotetraacetic acid (a chelator of calcium and magnesium). In contrast, this reduction was observed in plasma anticoagulated with [(ethylenebis(oxyethylenenitrilo)]tetraacetic acid (a chelator of calcium). The results demonstrate optimal conditions for activation of the alternative pathway of complement during absorption of rat plasma with Sepharose derivatives and suggest in vivo experiments to define the role of this pathway in inhibition of growth of mammary tumors.     

Ascitic complement system in ovarian cancer

Ovarian cancer spreads intraperitoneally and forms fluid, whereby the diagnosis and therapy often become delayed. As the complement (C) system may provide a cytotoxic effector arm for both immunological surveillance and mAb-therapy, we have characterised the C system in the intraperitoneal ascitic fluid (AF) from ovarian cancer patients. Most of the AF samples showed alternative and classical pathway haemolytic activity. The levels of C3 and C4 were similar to or in the lower normal range when compared to values in normal sera, respectively. However, elevated levels of C3a and soluble C5b-9 suggested C activation in vivo. Malignant cells isolated from the AF samples had surface deposits of C1q and C3 activation products, but not of C5b-9 (the membrane attack complex; MAC). Activation could have become initiated by anti-tumour cell antibodies that were detected in the AFs and/or by changes on tumour cell surfaces. The lack of MAC was probably due to the expression of C membrane regulators CD46, CD55 and CD59 on the tumour cells. Soluble forms of C1 inhibitor, CD59 and CD46, and the alternative pathway inhibitors factor H and FHL-1 were present in the AF at concentrations higher than in serum samples. Despite the presence of soluble C inhibitors it was possible to use AF as a C source in antibody-initiated killing of ovarian carcinoma cells. These results demonstrate that although the ovarian ascitic C system fails as an effective immunological surveillance mechanism, it could be utilised as an effector mechanism in therapy with intraperitoneally administrated mAbs, especially if the intrinsic C regulators are neutralised.     

Complement activation in vivo in cancer patients receiving C. parvum immunotherapy.

Serum complement levels were assayed in 26 patients with disseminated cancer, who received immunotherapy with infusion of C. parvum. Complement activation, indicated by the consumption of C3 or C4 or both, was found in 46% of the patients. Serum samples showed direct correlation between decreased C3 and conversion of C3 proactivator, whereas such conversion did not occur when C4 alone was decreased. It is concluded that the bypass (properdin) pathway was activated in patients in whom C3 consumption was detected, while the classical (C1) pathway was activated in the patients with C4 consumption unaccompanied by C3 decrease. Direct correlation was observed between delayed cutaneous hypersensitivity reactions to recall antigens and the incidence of C. parvum-associated complement activation.     

Isolation of total RNA from dermatophytes

We report a method for the preparation of total RNA from the anthropophilic dermatophyte Trichophyton rubrum. To generate large quantities of mycelia, the fungus was grown in liquid culture medium. The harvested mycelial mass was ground to a fine powder in liquid nitrogen and homogenized in guanidine isothiocyanate buffer followed by ultracentrifugation of the obtained suspension through a caesium chloride gradient. Analysis of the prepared RNA showed two prominent ribosomal RNA (rRNA) bands of about 3.36 and 1.82 kb. Northern blot hybridization with a beta-actin cDNA confirmed the high quality of the fungal mRNA. Successful isolation of RNA from two other dermatophyte species, namely Trichophyton mentagrophytes and Microsporum canis, demonstrated the general applicability of the described procedure.     

Serotherapy of primary rat mammary carcinoma: inhibition by ethylenedinitrilotetraacetic acid but not by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid

We reported inhibition of growth of primary rat mammary carcinomas after infusions of tumor-bearer plasma absorbed with Protein A-Sepharose or inactivated CNBr Sepharose. Absorbed plasmas were depleted of the third component of complement (C3) (other complement components defined similarly) and C5 but not C1, C4, or C2. These results suggested that activation of the alternative pathway of complement might be involved in the observed antitumor effects. To test this concept sera were treated with ethylenedinitrilotetraacetic acid or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid before absorption with Protein A-Sepharose. Ethylenedinitrilotetraacetic acid, by chelating calcium and magnesium, prevents activation of both the alternative and classical complement pathways. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid, by chelating calcium but not magnesium, permits activation of the alternative pathway but inhibits activation of the classical complement pathway. Sera in the presence or absence of chelating agent were absorbed with Protein A-Sepharose twice at room temperature. After absorption calcium was added to the sera. Rats were treated by i.v. injection of sera twice a week for 2 weeks. Measurements of tumor size were made weekly for 5-7 weeks and then tumor weight was determined. Groups were compared both for size of index and total tumors. The results can be summarized as follows: tumor-bearer sera before absorption did not inhibit the growth of rat primary mammary carcinomas; tumor-bearer sera after absorption with Protein A-Sepharose showed significant consumption of C3 and did inhibit tumor growth; tumor-bearer sera absorbed in the presence of ethylenedinitrilotetraacetic acid did not show a decrease in C3 functional activity and did not inhibit tumor growth; tumor-bearer sera absorbed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid did show a decrease in C3 functional activity and did inhibit tumor growth; sera from normal adult female rats after absorption with Protein A-Sepharose did inhibit tumor growth. The results are consistent with a role for the alternative pathway of complement in the inhibition of growth of rat primary mammary carcinomas observed after treatment with absorbed sera.     

Study on the effect of neem (Azadirachta indica A. juss) leaf extract on the growth of Aspergillus parasiticus and production of aflatoxin by it at different incubation times

Aflatoxins are secondary metabolites that are produced by toxigenic strains of some Aspergillus species on foods. Neem plant is a known inhibitor of aflatoxin production. We studied the effects of different concentrations of aqueous neem leaf extract on fungal growth and aflatoxin production by Aspergillus parasiticus (NRRL 2999) at different incubation times. The toxigenic fungus was cultured on sucrose low salts medium in the presence of various concentrations of extracts (0.2, 0.8, 3.12, 12.5 and 50% v/v). After shaking incubation of cultures for 2, 4, 6, 8, 10 and 12 days at 28 degrees C, the fungal mycelia was collected and processed for determination of dry weight. Mycelia and culture media were assayed by TLC method to detect aflatoxin B(1) (AFB(1)). The extracts did not have any obvious effect on fungal growth. AFB(1) production in the control samples increased to the maximum level on the 8th day. The inhibition of aflatoxin synthesis by plant extracts was found to be time- and dose-dependent. The maximum inhibitory effect was 80-90% in the presence of 50% concentration that when compared with control samples was significant (P < 0.05). AFB(1) secretion/production ratio in all of control and treated samples, other than 2nd day, approximately stayed and neem had no effect on it.     

Feline uveal melanomas induced with feline sarcoma virus: potential model of the human counterpart

Uveal melanomas were produced by injecting Gardner strain feline fibrosarcoma virus intraocularly into 10- to 15-day-old noninbred kittens. Tumors developed in about 90% of the cats' eyes receiving virus. Progressing tumors (62 eyes of 36 cats) began as small hyperpigmented lesions at the site of injection and grew to fill the anterior chamber by 3-5 months after infection. About 30% of cats with these tumors developed secondary tumors and died. Nonprogressive tumors characterized by flat, pigmented plaques on the iris at the site of injection developed in 25 eyes of 18 cats. These lesions did not enlarge except in proportion to the growth of the eye. Tumors were composed of pigmented spindle cells, pigmented epithelioid cells, and nonpigmented spindle cells. The cells could be grown for 5-8 passages in vitro. One culture assumed a transformed morphology and grew in noninbred athymic nu/nu mice. The lesions resembled human spindle cell melanomas.     

Restoration of depressed immune responses by PSK in C3H/He mice bearing the syngeneic X5563 tumor

PSK is a protein-bound polysaccharide prepared from cultured mycelium of Coriolus versicolor. The effects of PSK on immunologic responsiveness were investigated in C3H/He mice bearing syngeneic X5563 tumor. The results were as follows. elayed foot pad reaction and antibody-forming capacity to sheep erythrocytes were depressed in tumor bearing mice, and such depression was prevented by oral or intraperitoneal administration of PSK. In vitro cytotoxic activity of splenic lymphocytes against the tumor was augmented by PSK administration. Antitumor effect was augmented by combination of PSK and X-irradiation. Delayed foot pad reaction to sheep erythrocytes was suppressed in normal C3H/He mice given immunosuppressive substance obtained from tumor-bearing mice, and the depressed reaction recovered to the normal level following PSK administration. These results show that PSK is effective in the syngeneic murine tumor system.     

Two new media Pinus halepensis seed agar and blackberry agar for rapid identification of Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast‐like fungus that causes life‐threatening infections, particularly in immunocompromised patients. The formation of brown pigment on many media described in the literature, such as that in Niger seed (Guizotia abyssinica) agar, has been used to identify C. neoformans. The present study compares melanin production by clinical and environmental isolates of C. neoformans and other medically important yeast on two new media, Pinus halepensis seed (PHS) agar and blackberry (BlaB) agar, and the classic medium Niger seed agar. Results obtained after the culture of 46 strains of C. neoformans, for 4, 24 and 48 h at 37 °C on these three media, showed that at 24 h, 100% of strains were pigmented on BlaB agar, 91.3% on PHS agar but only 34.8% on Niger seed agar. In conclusion, PHS and BlaB agar are two interesting new media for the rapid identification of C. neoformans isolates.     

Role of uromodulin and complement activation in the progression of kidney disease

Uromodulin (UMOD) is a glycoprotein that is selectively expressed on the epithelial cells of the thick ascending limb of Henle''s loop and the early distal renal tubule. The present study aimed to investigate whether UMOD was associated with complement activation in patients with renal diseases. In addition, its biological function was examined in vitro. The expression levels of UMOD and complement components, including C1q, C3, C4 and C3a, and membrane attack complex (MAC) in the plasma of patients with IgA nephropathy (IgAN; n=58) and lupus nephritis (LN; n=36) were detected using ELISA, which was used to determine the association between UMOD expression and complement components. In addition, a simulated hypoxia-reoxygenation (H/R) model was used to stimulate UMOD expression in mouse inner medullary collecting duct cells. Additionally, the association between UMOD expression and complement components C1q and C3d at the cellular level was identified using western blotting and immunofluorescence, respectively. It was revealed that the plasma UMOD concentration was significantly decreased in patients with IgAN and LN compared with in healthy controls, and the levels of C3a and MAC were significantly increased in the plasma of patients with IgAN and LN. Furthermore, the plasma levels of C1q, C3 and C4 in patients with LN, but not in patients with IgAN, were significantly decreased compared with in healthy controls. The plasma levels of UMOD were negatively correlated with the plasma C3a and MAC concentrations. However, the plasma levels of UMOD were significantly and positively correlated with the plasma C1q concentration, but not with that of C3 and C4. It was identified that UMOD expression started to increase after 1 h of simulated H/R, and continued to increase at 6 and 12 h. In addition, cells with lower UMOD expression had higher C3d expression in vitro. Collectively, the present results suggested that UMOD was associated with severe complement activation and may be involved in complement-mediated immune protection by inhibiting complement activation in renal disease.     

Candida ciferrii: clinical and microbiological features of an emerging pathogen

The authors report six cases of toenail onyxis due to an unusual yeast species, Candida ciferrii. For half of these cases, direct microscopical examination showed the presence of blastospores and pseudo-to-true mycelium, demonstrating the parasitic transition of the fungus. In light of the literature and of their own experience, the authors suggest that C. ciferrii could be an etiological agent of onychomycosis, particularly for elderly patients with extensive trophic disorders.     

Binding of the food mutagen PhIP in pigmented tissues of mice.

The distribution of the 14C-labelled food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the tissues of C57B1/6 and NMRI mice was studied. The results showed a high and selective binding of radioactivity in the pigment epithelium of the eye and in the fur following a single dose (0.3-4 mg/kg) of [14C]PhIP in the pigmented C57B1/6 mice whereas no such localization of radioactivity was present in the albino NMRI mice. A low but selective covalent binding of radioactivity was observed in the liver, inner cortex of the kidney and in the tracheal mucosa of [14C]PhIP-injected mice. PhIP was firmly bound to synthetic melanin pigment in vitro; only 3% was released by extraction with a phosphate buffer (pH 7) whereas 72% was released by extraction by methanol:conc. NH3 (15:1). Three hours to 7 days following a single injection of [14C]PhIP in C57B1/6 mice the radioactivity in the eye was 3- to 6-fold higher than that in the liver or kidney. Almost 60% of the radioactive material present in the pigmented epithelium of the eye 3 and 24 h following injection could be extracted by basic methanol and identified as unchanged PhIP. The residual radioactivity in the pigmented epithelium of the eyes may represent a covalent binding of [14C]PhIP metabolites to cellular constituents or to a basic methanol-resistant binding of [14C]PhIP to melanin. The results indicate that pigmented tissues may be potential target tissues for the toxic effects of PhIP and suggest that the use of hair for biological monitoring of PhIP should be examined.     

Macrophage accumulation in primary and transplanted tumors growing in C5-deficient B10.D2/oSn mice

The hypothesis was tested that the fifth component of complement (C5) was required for the accumulation of macrophages at the site of tumor growth. This was based on the assumption that the cleavage product of C5, C5a, attracts peripheral blood monocytes along a chemotactic gradient. Methylcholanthrene-induced primary and transplanted tumors were grown in the genetically C5-deficient B10.D2/oSn strain and the C5-positive B10.D2/nSn strain and their macrophage content assessed after enzymic disaggregation of the tumors. The overall data indicated that there were no significant differences in the two strains since macrophages accumulated in the tumors irrespective of the presence of C5 or its cleavage product C5a.     

A comparison between the effect of step-down heating in a tumour and a normal tissue in vivo

A comparison between the effect of step-down heating (SDH) obtained in a C3H mammary carcinoma grown in the feet of CDFl mice and the skin of normal CDFl feet is presented. Water-bath heating was used, and SDH was obtained by giving a 44.7°C/10 min treatment followed by heating at 42.2°C for variable times. Single heating at 42.2°C and step-up heating (SUH), i.e. 42.2°C followed by 44.7°C/10 min, were used as controls. The endpoint was the heating time at 42.2°C to obtain either a definite tumour growth time (TGT50) or a specific skin score level (RD50) in 50% of the animals. The effect of SDH and SUH was quantified by the step-down ratio (SDR), calculated as the ratio of the heating times at 42–2°C to obtain the specific endpoint. In both assays the effect of SDH was seen as a significant left shift of the SDH dose-response curve compared to the curve for single heating and SUH. For the comparison of the tumour and the normal tissue response, damage levels with comparable heating times for single heating were used. The therapeutic effect was then investigated by calculating the therapeutic gain factor (TGF), where TGF = SDR(tumour)/SDR(normal tissue). Neither SUH nor SDH gave a TGF significantly different from 1. The results suggest that SDH may be used clinically to shorten the heating time without decreasing the therapeutic effect.