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Female BALB/c mice were immunized with a whole-hyphal-cell extract obtained from Candida albicans wild-type strain 4918 grown in Lee medium. Monoclonal antibody (MAb)-producing hybridomas were prepared by fusing immune splenocytes with NS-1 myeloma cells. One of the hybrid cell clones (1.183) secreted an immunoglobulin G1 antibody that reacted with C. albicans hyphae in an indirect immunofluorescence assay but not with yeast cells and pseudohyphal segments directly originating from parent blastoconidia. In the same assay eight of nine recent clinical C. albicans isolates and Candida stellatoidea tested positive for hyphal cell-specific reactivity with MAb 1.183. The recognized antigen on hyphal cells was sensitive to heat treatment, beta-mercaptoethanol reduction, and proteolysis with pronase, trypsin, and subtilisin. Western blot (immunoblot) analysis of hyphal whole-cell and dithiothreitol extracts with MAb 1.183 revealed two major proteins with approximate molecular masses of 55 and 60 kilodaltons (kDa) under reducing conditions. Endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment reduced the molecular mass of the 60-kDa protein slightly but did not affect recognition by MAb 1.183, whereas peptide:N-glycosidase F (N-glycanase) had no effect on either protein. When exponentially growing yeast cells were treated sequentially with EDTA, beta-mercaptoethanol, and Zymolase to form protoplasts, a specific immunofluorescence signal was obtained with MAb 1.183. In a Western blot, MAb 1.183 showed reactivity with a 20-kDa protein in the sodium dodecyl sulfate extract from protoplasts, whereas no reactivity was found with cell wall material obtained from yeast cells. In summary, these experiments indicated that specific cell surface components from C. albicans hyphae are related to antigens which are present in yeast cells but are not detectable on the surface of the latter.

The expression of the Candida albicans complement-binding C3d protein (MP60) was investigated both in vitro and in vivo by immunogold labelling and electron microscopy. In vivo expression was determined in a rat vaginitis model. Reactivity of in vitro-grown cells to an anti-MP60 rabbit serum was associated with both cytoplasmic and cell wall sites. Immunostaining in the cell wall of both yeast and hyphae was most concentrated in the inner, electron-lucid layer. Immunogold stained preparations of C. albicans from vaginal smears of infected animals also showed intense localization of the MP60 in the inner cell wall, plasma membrane. However, immunogold label was also intense at the cell surface in these samples, mostly in the area of close adherence with the keratinocytes of the vaginal epithelia. These observations indicate that MP60 is expressed both in vitro and in vivo, but to a different degree in the different cell wall layers.

We have identified and analysed a putative response regulator two-component gene (CaSSK1) from Candida albicans and its encoding protein (CaSsk1p). CaSSK1 has an open reading frame of 2022 bp. In the promotor region of CaSSK1 a short sequence is found that matches the consensus sequence of the stress response elements (STRE) from Saccharomyces cerevisiae. CaSSK1 is located on chromosome 1 and is expressed in either yeast or mycelial phases of C. albicans. CaSSK1 encodes a 674 amino acid protein (CaSsk1p) with an estimated molecular mass of 73.5 kDa and a basic isoelectric point (pI 9.5). It has a tripeptide (NKA) located in its C-terminus, which resembles the peroxisomal signalling target type 1 sequence (PST1) of most of the peroxisomal matrix proteins. A homology search of CaSsk1p with other proteins in databases showed that the C-terminus of CaSsk1p exhibits the greatest similarity with the C-terminus of Ssk1p and Mcs4 from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The response regulator domain of CaSsk1p contains the motifs that are characteristic of all response regulators, including the conserved aspartate and lysine residues as well as the putative aspartate, which is phosphorylated by a phosphohistidine residue. Finally, in spite of the structural similarities among CaSsk1p, Ssk1p and Mcs4, CaSsk1p does not seem to exhibit functional homology with these proteins.

We have cloned and analysed the sequence of a putative histidine kinase, two-component gene (CaHK1) from Candida albicans. This gene encodes a 2471 amino acid protein (Cahk1p) with an estimated molecular mass of 281.8 kDa. A homology search of Cahk1p with other proteins in the databases showed that Cahk1p exhibits the greatest homology at its C-terminus with both the sensor and regulator components of prokaryotic and eukaryotic two-component histidine kinases. A further analysis of this homology showed that the Cahk1p possessed both sensor and regulator domains in the same polypeptide. Also, Cahk1p is likely to be a soluble protein. The sensor kinase domain of Cahk1p contains conserved motifs that are characteristic of all histidine kinase proteins, including the putative histidine which is believed to be autophosphorylated during activation, ATP binding motifs and others (F- and N-motifs), with unknown function. The Cahk1p regulator domain also contains conserved aspartate and lysine residues and the putative aspartate, which is secondarily phosphorylated by the autophosphorylated histidine. Finally, according to the codon usage frequency of the CaHK1 gene in comparison with other genes from C. albicans, there would appear to be a low level of expression of the gene.

Although reduced by technology, antibiotics and surgical technique, spinal infection from surgery remains a recognizable risk. The rate of infection in spinal surgery is reviewed. Identification of risk factors are important in preoperative planning. Preoperative risk factors for postoperative spinal infection include obesity and smoking. Attention to sterility and efficient technique can reduce potential wound contamination intraoperatively. Excessive wound drainage and seroma formation should warn of a potential wound infection.

The movement towards managed care has raised the awareness of health care costs in today's society. The additional expense involved in treating patients with deep postoperative spinal infections after lower back fusion increases the total cost of care more than four times. Three areas of greatest increase in cost are room and board, pharmacy and laboratory charges. Decreasing the expense of this complication can best be effected through use of home nursing care, choice, and duration of antibiotic treatment and prudent laboratory testing.

Hematogenous spread is the most common cause for vertebral osteomyelitis. S. aureus is the most common organism in pyogenic vertebral osteomyelitis. Hematogenous osteomyelitis is common among diabetics and intravenous drug abusers. Tuberculous spondylitis remains common worldwide. In general, vertebral body infections not responding to antibiotic treatment and those creating unacceptable deformity or neurologic compromise require debridement via an anterior approach with strut grafting. Posterior infections are almost always postsurgical and require posterior irrigation and debridement in addition to antibiotics. Abscesses within the canal require antibiotics and surgical debridement especially when neurologic symptoms are present. Infections within the canal are approached posteriorly unless the pathology involves the anterior spine. Use of metal fixation within the site of an adequately debrided spinal infection is controversial but necessary on rare occasions. Posterior fixation for anterior infections is preferred. Much has been written about spinal infections and their treatment. Landmark articles and additional comprehensive sources on spinal infections have been included in the references.

The importance of outcome studies in patients undergoing spinal surgery is discussed. There are numerous questionnaires used to assess quality of life results in orthopedic patients. This suggests disagreement and difficulty in the assessing and comparing outcomes. Several of these instruments for assessing outcome are reviewed. Postoperative spinal infection can prolong a patients recovery from spinal surgery and exacerbate symptoms of lower back pain. A review of the authors' patients at 3 years follow-up suggests that outcome with postoperative infections was similar to other patients with low back pain undergoing fusion without postoperative infectious complications.

We have isolated a 3.7 kb EcoR1 fragment from a genomic library of Candida albicans which displayed a 65% level of identity with the PRS gene family (PRS) of Saccharomyces cerevisiae. The PRS gene encodes a phosphoribosylpyrophosphate (PRPP) synthetase of S. cerevisiae, which catalyses the synthesis of purines, pyrimidines, and amino acids such as histidine and tryptophan. By Northern analyses, we observed that the entire 3.7 kb EcoR1 fragment as well as 1.1 kb KpnI-SacI internal fragment of the 3.7 kb EcoR1 fragment hybridized to the same 1.4 kb transcript. An internal 2.6 kb KpnI fragment was subcloned and sequenced. A deduced sequence of 321 amino acids representing a polypeptide of 35.2 kDa was determined. A FASTA search indicated that the C. albicans PRS (Ca PRS1) had an overall homology at the amino acid level of 91% with the S. cerevisiae PRS3. Putative transcriptional start and termination sequences as well as a cation-binding, PRPP synthetase signature sequence were identified. Ca PRS1 was localized to chromosome 2 of the C. albicans genome. Low stringency hybridizations indicates that the organism may possess multiple PRS genes. The function of these genes in nitrogen signaling is discussed.

A case presentation of shoulder dislocation with ipsilateral humeral shaft fracture is presented along with a review of the literature regarding nine reported cases. In the current case, closed treatment of the humeral shaft fracture was undertaken with an unsuccessful attempt at closed reduction of the anterior shoulder dislocation, resulting in a radial nerve deficit. Successful treatment required open reduction of the humeral fracture with compression plating followed by closed reduction of the shoulder dislocation. We compare this treatment (with outcome) to other methods of treatment.

e13570 Background: Namitecan (N) is a topoisomerase I inhibitor with superior antitumor activity (especially in squamous cell ca., SCC) and a more favourable safety profile than irinotecan and topo...

Four clones of the yeast Candida albicans, isolated on the basis of their tolerance to clotrimazole, were compared with their parental strains in terms of growth, morphology, virulence, and cell surface complement receptor activity. In a newly described synthetic medium, these clones, designated C1, C2, N, and P, produced germ tubes or pseudohyphae, but no true hyphae, in a pattern which was specific for each strain. The growth of each clone at 37 degrees C, under conditions which favor the filamentous growth form of the organism, was equal to that of the parental strain (H12). The pathogenicity of each clone was tested in an intravenous mouse model. None of the mice infected with the tolerant clones but all of the mice infected with H12 developed severe renal candidiasis after infection with 1.4 x 10(6) to 2.0 x 10(6) CFU/ml. The number of CFU of each clone from the mouse kidney was reduced about 3 or 4 orders of magnitude in comparison with the wild type. As a correlate, we measured the complement receptor activity (CR2 and CR3) of each clone. The C3 ligands, iC3b and C3d, were conjugated to sheep erythrocytes (E) sensitized with antibody (A) to the erythrocytes (EA). We found that all tolerant clones showed reduced recognition of C3d-bearing sheep erythrocytes (EAC3d) in rosetting assays. Clone P showed more than an 80% reduction in rosetting of EAC3d in comparison with H12 cells. In contrast, recognition of iC3b (EAiC3b) by each of the clones was similar to that by H12 cells. When dithiothreitol extracts of clone P and H12 were compared by immunoblot, both quantitative and qualitative differences in reactivities were observed with antibodies specific for the Candida C3d receptor and with antiserum from a patient with chronic mucocutaneous candidiasis.

The interaction of C. albicans with host cells has been shown to be quite complex. At least three recognition systems have been described, and, probably additional ones exist. The recognition systems are classified by the type of host cell, the growth form of the organism and the nature of the interaction at the molecular level, i.e., protein-protein or protein-oligosaccharide. It would appear that C. albicans has at least two distinct mannoprotein adhesins. One resembles a lectin in that it recognizes host cell glycosides containing fucose, and with some strains of C. albicans, N-acetylglucosamine. The second adhesin has properties similar to the integrin or transmembrane glycoproteins such as the CR3 (complement receptor 3). Still to be resolved is the characterization of the lectin-like adhesin as well as the relationship of the CR2-like activity to the CR3 of Candida. A third adhesin which is a mannan polysaccharide has been suggested as a recognition molecule in the adherence of the organism to buccal epithelial cells.

The complement conversion product C3d binds to a receptor on the cell surface of Candida albicans. While the function of this receptor is still uncertain, we investigated whether it is expressed during a murine infection. Rabbit antiserum raised against purified receptor was used in conjunction with immunofluorescence microscopy and immunocolloidal gold electron microscopy to examine kidney tissue and peritoneal lavages from infected mice for receptor expression by C. albicans in vivo. Specificity of the antiserum was indicated by reactivity with purified receptor (55 to 60 kDa) and with a protein of similar molecular mass from whole hyphal extracts in Western blots (immunoblots). In vitro analysis by immunofluorescence microscopy showed that the antiserum reacted with both yeast and pseudohyphal forms of the organism, but reactivity was strongest with pseudohyphae. Immunocolloidal gold electron microscopy of fungal cells from peritoneal lavages revealed intense staining of mother cells of germinative forms, germ tubes, and pseudohyphae. Staining of the mother cells was heaviest at the innermost layers of the cell wall but only scant on the cell surface. In contrast, staining was observed throughout the cell walls of germ tubes and pseudohyphae. In kidney, expression of the C3d receptor was found primarily on the cell walls of hyphae and pseudohyphae, although some staining was observed in the cytoplasm. These data support that the C3d receptor of C. albicans is expressed in vivo.

Various types of mammalian cells possess receptors for the complement C3 conversion products C3b, iC3b and C3d. In the case of C3b and iC3b, these products act as ligands as they are generated on the surface of microorganisms opsonized with complement (Ross, 1986). Phagocytic cells use receptors which recognize these ligands as a first step in the process of eliminating opsonized organisms from circulation. The receptor for C3b and iC3b are referred to as complement receptor 1 (CR1) and complement receptor 3 (CR3), respectively (Ross, 1986). They are not found exclusively on phagocytic cells and have been described from erythrocytes and lymphocytic cells (Ross, 1986). The CR3 receptor is a glycoprotein with an α chain of 165 kDa molecular mass and a 95 kDa β-chain. It is a member of a family of receptors referred to as “integrins” which have a common β-chain and a variable α-chain. These receptors are transmembrane proteins and, in addition to their role in phagocytosis (as, for example, the CR3), also confer adhesive properties and are involved in homing mechanisms for mammalian cells (Stoolman, 1989).

Species of the Pythiaceae, consisting of the genera Pythium and Phytophthora, have been shown to require the presence of exogenous sterols for reproduction and/or growth (3, 5, 6, 7, 8, 10, 15). Schlosser and Gottlieb (14) have shown that when species of Pythium bind exogenous sterols, they become sensitive to the polyene antibiotic filipin. Such action indicates localization of sterol at the cell membrane since polyene antibiotics bind with sterols at that site. These workers also report (15) that Pythium ultimum Trow in the presence of cholesterol utilized labeled glucose and released 14C02 at a faster rate than did the sterol-free mycelium. Thus, sterols, which appear to be localized at the cell membrane, may be involved in some manner in the metabolism or uptake of carbohydrates. Little information exists concerning the effects of sterols on enzyme activity of Phytophthora spp. Since sterols have been implicated in the respiratory process (15, 16), three key respiratory-associated enzymes, aldolase, L-glutamic acid-oxaloacetic acid transaminase (GOT), and glucose-6-phosphate dehydrogenase (GPDH) were chosen for study in cultures of Phytophthora cactorum (Leb. & Cohn) Schroet. grown with and without /3-sitosterol. The isolate of Phytophthora cactorum used in this study, N-94, was

Normal rabbit alveolar macrophages were infected in vitro with Candida albicans. Early after infection, germ tube formation of phagocytized C. albicans was inhibited in contrast to extracellular (nonphagocytized) C. albicans. Over and 8-h period, plate counts of C. albicans incubated with alveolar macrophages revealed a decrease in colony-forming units in contrast to C. albicans alone. In addition, an assay was developed which specifically measured C. albicans [3H]leucine incorporation in the presence of alveolar macrophages. Using this assay, we observed a 71 to 93% inhibition of macromolecular synthesis in C. albicans when incubated with alveolar macrophages. Autoradiographic studies showed that the inhibition of leucine incorporation was restricted to the ingested Candida.

An alkali-soluble extract from a cell wall preparation of Candida albicans was conjugated to sheep erythrocytes (SRBC) by using periodate oxidation or concanavalin A. Conjugated SRBC readily attached to a fibrin-platelet matrix, whereas nonconjugated SRBC did not. To determine the active component which promoted attachment of SRBC, the alkali-soluble fraction was treated with alpha-mannosidase, pronase, or glusulase or chemically degraded by acetolysis. The treated extract was then reconjugated with SRBC, and attachment was measured. When treated with alpha-mannosidase or degraded by acetolysis, the alkali-soluble extract failed to promote the adherence of SRBC to the fibrin-platelet matrix. Pronase- or glusulase-digested extract promoted attachment equally as well as untreated controls. In addition, when preabsorbed with antiserum to whole cells of C. albicans, the alkali extract abrogated the inhibition of adherence by antiserum, thus indicating its antigenicity. The extract consisted primarily of polysaccharide (72%) and contained a small amount of protein (less than 1%). Mannose and glucose (ratio, 3:1) were detected by gas-liquid chromatography. These data indicate that cell surface mannan may play an important role in the adherence of C. albicans to the fibrin-platelet matrices which form in vivo on the endocardium of heart valves.

The interaction of human platelets and Candida albicans was studied. Platelet-rich plasma was obtained from freshly drawn blood or outdated platelet concentrates. From the platelet-rich plasma, a platelet extract was derived which stimulated germ tube formation by C. albicans when incubated with yeast cells at 37 degrees C. The active component(s) was heat stable, trypsin sensitive, and ribonuclease and deoxyribonuclease insensitive, and possessed cationic properties since it readily attached to carboxymethyl-Sephadex. The active component(s) seemed to bind to heparin also, since germ tube-promoting activity was eluted from a heparin-cyanogen bromide-activated Sepharose 4B column. In addition, platelet-derived growth factor (Collaborative Research, Inc.) stimulated germination when incubated with low amounts (0.4% final concentration) of bovine calf serum. The aggregation of platelets, prepared as platelet-rich plasma by C. albicans cell wall or alkali-extracted cell wall fractions, was also studied. Aggregation of platelets was observed when cell wall or cell wall fractions were incubated with platelet-poor plasma at 37 degrees C for 20 min and then added to platelet-rich plasma. The component of platelet-poor plasma which promoted aggregation of platelets by C. albicans cell wall or alkali-extracted fractions was inactivated at 56 degrees C (30 min) and by cobra venom factor, indicating a role for the alternate complement pathway in the aggregation response.

Pseudohyphal but not yeast forms of Candida albicans possess both iC3b and C3d receptors, as determined by rosetting with erythrocytes carrying iC3b (EAC3bi) or C3d (EAC3d). Rosetting with EAC3d was markedly reduced when pseudohyphae were heat killed or treated with trypsin or pronase but was not inhibited by several saccharides or aminosaccharides, including alpha-methyl-D-mannoside, or by pretreatment of pseudohyphae with concanavalin A. However, mannoproteins obtained by concanavalin A affinity chromatography of whole pseudohyphal extracts inhibited the attachment of EAC3d to C. albicans, whereas soluble (nonmannosylated) proteins were less active. Thus, although the C3d receptors appeared to be glycosylated, the oligosaccharide component of the receptor was apparently not involved in the recognition of C3d. To isolate these receptors, whole-cell extracts were separated by DEAE-Trisacryl chromatography. Fractions that inhibited rosetting were pooled and affinity purified by C3d-Thiol-Sepharose chromatography. The eluate from this affinity column inhibited attachment of C. albicans to EAC3d. Monoclonal antibodies to C. albicans were prepared, and three of these antibodies blocked rosetting. Western blotting (immunoblotting) with these antibodies indicated the presence of 62- and 70-kilodalton receptors for C3d in the extracts purified by C3d affinity chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The adherence of Candida albicans to a fibrin-platelet matrix formed in vitro was studied. Platelet-rich plasma obtained from rabbits was incubated with thrombin and CaCl2 to form a clot in tissue culture dishes. Such clots were then infected with 3 x 10(7) C. albicans cells per 0.3 ml prelabeled with [U14C]-glucose, and the percent adherence was measured after 30 min of incubation by counting the radioactivity in saline washes of the clot as well as a streptokinase-streptodornase digest of the corresponding clot. Heat- and formaldehyde-killed cells did not adhere as well as viable cells. Pretreatment of C. albicans with trypsin, chymotrypsin, and pronase reduced adherence to the clots. Normal rabbit serum and anti-Candida antiserum also inhibited adherence 40 and 100%, respectively, Diethylaminoethyl-purified anti-Candida gamma globulin (1:8) completely inhibited adherence, whereas purified normal serum gamma globulin did not. Several Candida spp. and Saccharomyces cerevisiae showed differences in their ability to adhere to clots. C. albicans and C. stellatoidea presented the highest adherence, whereas C. krusei, C. guilliermondii, and S. cerevisiae adhered less readily. Other species were intermediate in their ability to adhere.

Endocarditis caused by Candida albicans was induced in rabbits after insertion of a catheter across the aortic valve. The mean survival time of 34 rabbits was 26 days. Only 7% of temperature recordings taken were elevated. Candida was recovered from only 9% of blood cultures taken. Precipitating and agglutinating serum antibody was detected after 12 days of infection. Antibody titers rose progressively until death in rabbits with endocarditis, whereas titers peaked early and subsequently decreased in animals that received an intravenous injection of C. albicans without precatheterization. Three groups of rabbits were treated for 6 days with amphotericin B, 5-fluorocytosine, or the two durgs in combination. Amphotericin B alone reduced the mean titer of organisms from log10 8.79 +/- 1.46 to log 10 3.1 +/- 1.9 colony-forming units/g. 5-Fluorocytosine was less effective (mean titer after 6 days of therapy was log10 7.4 +/- 0.33 colony-forming units/g). The addition of 5-fluorocytosine to amphotericin B did not increase the rate at which Candida cells were eradicated from the vegetations. These in vivo results corrleated with the failure to demonstrate an increased rate of fungicidal activity in vitro with the two drugs.

Lysosomal-rich fractions, obtained from normal rabbit alveolar macrophages, were extracted and tested for their effects on Candida albicans. The uptake and incorporation of various compounds (amino acids, uridine, 2-deoxy-D-glucose, and Rb+) by C. albicans were measured in the presence and absence of extract. These studies demonstrated that the extract had a specific effect on the uptake of certain amino acids by C. albicans. Of the amino acids tested, the uptake of methionine valine, lysine, phenylalanine, and leucine was drastically reduced in the presence of extract, whereas proline and glutamic acid uptake was unaffected. Those amino acids whose uptake was inhibited have been shown to be transported in other yeasts by a general amino acid permease. The existence of a general amino acid permease in C. albicans is compatible with our data. Additionally, the extract had no effect on the uptake of uridine, 2-deoxy-D-glucose, and Rb+. Leakage of 86Rb by C. albicans was detected in the presence of the extract. Viability of Candida, as measured by colony-forming ability, decreased after a 16-h incubation of C. albicans with the extract.