Search

The vacuole has crucial roles in stress resistance and adaptation of the fungal cell. Furthermore, in Candida albicans it has been observed to undergo dramatic expansion during the initiation of hyphal growth, to produce highly "vacuolated" subapical compartments. We hypothesized that these functions may be crucial for survival within the host and tissue-invasive hyphal growth. We also considered the role of the late endosome or prevacuole compartment (PVC), a distinct organelle involved in vacuolar and endocytic trafficking. We identified two Rab GTPases, encoded by VPS21 and YPT72, required for trafficking through the PVC and vacuole biogenesis, respectively. Deletion of VPS21 or YPT72 led to mild sensitivities to some cellular stresses. However, deletion of both genes resulted in a synthetic phenotype with severe sensitivity to cellular stress and impaired growth. Both the vps21Delta and ypt72Delta mutants had defects in filamentous growth, while the double mutant was completely deficient in polarized growth. The defects in hyphal growth were not suppressed by an "active" RIM101 allele or loss of the hyphal repressor encoded by TUP1. In addition, both single mutants had significant attenuation in a mouse model of hematogenously disseminated candidiasis, while the double mutant was rapidly cleared. Histological examination confirmed that the vps21Delta and ypt72Delta mutants are deficient in hyphal growth in vivo. We suggest that the PVC and vacuole are required on two levels during C. albicans infection: (i) stress resistance functions required for survival within tissue and (ii) a role in filamentous growth which may aid host tissue invasion.

The ability of the pathogenic fungus Candida albicans to cause disease requires rapid adaptation to changes in the host environment and to an evolving host immune response. The identification of 'virulence factors' using in vitro characterization of mutant strains has traditionally relied on a common set of phenotypic and biochemical assays (most often performed at 30 degrees C) and the subsequent correlation with their corresponding virulence in mouse models of disease. Utilizing a panel of isogenic mutants for the multifunctional signal-modulating 14-3-3 protein (Bmh1p), we have found that specific mutations affect a variety of different pathways currently associated with virulence, including those involved with the formation of filaments, as well as interaction with host immune cells. Surprisingly, our studies revealed that deficiencies in many of these pathways do not always correlate with virulence in a mouse model of disseminated infection. Mutations within the binding pocket of Bmh1p that affect the ability of the protein to efficiently bind ligand had varying effects on the results of a number of in vitro and in vivo assays. The capability, in vitro, to filament in embedment conditions, and to filament and form chlamydospores under microaerophilic conditions on cornmeal agar, does not correlate with virulence. It is likely that only a subset of hyphal signalling pathways is actually required for the establishment of infection in the disseminated mouse model. Most importantly, our results suggest that the delayed onset of log-phase [corrected] growth in vitro at 37 degrees C, and not at 30 degrees C, results in an inability of these mutants to rapidly adjust to environmental changes in vivo and may be responsible for their increased clearance and reduced virulence. It is critical, therefore, that future in vitro studies of putative virulence factors in C. albicans include careful characterization at physiological temperatures.

Autophagy is a major cellular process that facilitates the bulk degradation of eukaryotic macromolecules and organelles, through degradation within the lysosomal/vacuole compartment. This has been demonstrated to influence a diverse array of eukaryotic cell functions including adaptation, differentiation and developmental programmes. For example, in Saccharomyces cerevisiae autophagy is required for sporulation and survival of nitrogen starvation. The opportunistic pathogen Candida albicans has the ability to colonize and cause disease within a diverse range of mammalian host sites. The ability to adapt and differentiate within the host is liable to be critical for host colonization and infection. Previous results indicated that the vacuole plays an important role in C. albicans adaptation to stress, differentiation, and survival within and injury of host cells. In this study the importance of vacuole-mediated degradation through the process of autophagy was investigated. This involved identification and deletion of ATG9, a C. albicans gene required for autophagy. The deletion strain was blocked in autophagy and the closely related cytoplasm to vacuole (cvt) trafficking pathway. This resulted in sensitivity to nitrogen starvation, but no defects in growth rate, vacuole morphology or resistance to other stresses. This indicates that the mutant has specific defects in autophagy/cvt trafficking. Given the importance of autophagy in the development and differentiation of other eukaryotes, it was surprising to find that the atg9Delta mutant was unaffected in either yeast-hypha or chlamydospore differentiation. Furthermore, the atg9Delta mutant survived within and killed a mouse macrophage-like cell line as efficiently as control strains. The data suggest that autophagy plays little or no role in C. albicans differentiation or during interaction with host cells.

Yeast-hypha differentiation is believed to be necessary for the normal progression of Candida albicans infections. The emergence and extension of a germ tube from a parental yeast cell are accompanied by dynamic changes in vacuole size and morphology. Although vacuolar function is required during this process, it is unclear if it is vacuolar expansion or some other vacuolar function that is important. We previously described a C. albicans vps11Delta mutant which lacked a recognizable vacuole compartment and with defects in multiple vacuolar functions. These include sensitivities to stress, reduced proteolytic activities, and severe defects in filamentation. Herein we utilize a partially functional VPS11 allele (vps11hr) to help define which vacuolar functions are required for differentiation and which influence interaction with macrophages. Mutant strains harboring this allele are not osmotically or temperature sensitive and have normal levels of secreted aspartyl protease and carboxypeptidase Y activity but have a fragmented vacuole morphology. Moreover, this mutant is defective in filamentation, suggesting that the major role the vacuole plays in yeast-hypha differentiation may relate directly to its morphology. The results of this study support the hypothesis that vacuole expansion is required during germ tube emergence. Both vps11 mutants were severely attenuated in their ability to kill a macrophage cell line. The viability of the vps11delta mutant was significantly reduced during macrophage interaction compared to that in the control strains, while the vps11hr mutant was unaffected. This implies some vacuolar functions are required for Candida survival within the macrophage, while additional vacuolar functions are required to inflict injury on the macrophage.

A method for the analysis of Candida albicans gene function, which involves random mutagenesis of the open reading frame, is described. This method is especially suited for the study of essential and multi-functional genes, with several advantages over regulatable promoters more commonly used to study essential gene function. These advantages include expression from the endogenous promoter, which should yield a more appropriate transcript expression and abrogate the need for shifts in carbon or amino acid sources necessary with the use of regulatable promoters. Furthermore, there is potential for isolating individual functions of multi-functional genes. To verify this experimental approach, we randomly mutated the essential C. albicans gene, BMH1. The resulting "pool" of putative mutant alleles was then introduced into a BMH1/bmh1Delta strain of C. albicans, such that only the mutagenized BMH1 sequences could be expressed. Transformants were screened for rapamycin sensitivity, defects in filamentation on M199 agar, and growth at 42 degrees C. In this way, we identified six non-lethal mutant alleles of BMH1 with altered amino acid sequences. Further phenotypic analysis of these mutant strains enabled us to segregate individual functions of C. albicans BMH1. The relative merits of Escherichia coli versus PCR-mediated mutagenesis are discussed.

Objective: The purpose of this study was to compare cost and efficacy of surgical closure of patent ductus arteriosus using new critical pathway methods with outpatient transcatheter coil occlusion of patent ductus arteriosus., Methods: Surgical techniques included a transaxillary, muscle-sparing thoracotomy, triple ligation of the patent ductus arteriosus, no chest tube, and discharge from the hospital within 24 hours. Transcatheter coil occlusion of patent ductus arteriosus was done as an outpatient procedure. Costs were compared with inclusion of all hospital and professional charges., Results: From July 1994 until March 1996, 20 patients underwent coil occlusion of patent ductus arteriosus and 20 patients underwent surgical closure of patent ductus arteriosus. Duration of hospitalization was significantly less for the patients receiving coil occlusion (11 +/- 6 hours) as compared with that for the patients having surgical ligation (28 +/- 7 hours, p < 0.05). Total charges were similar for surgical ligation ($7101 +/- $408) as compared with those for coil occlusion ($7104 +/- $886, p > 0.05). Morbidity in coil occlusion included inability to occlude the patent ductus arteriosus in two patients (2/20, 10%) and residual patency in two patients (2/18, 11%). Morbidity in the surgical group included nausea and vomiting necessitating hospitalization for more than 36 hours in one patient (1/20, 5%), transient left recurrent laryngeal nerve palsy in one (1/20, 5%), and pneumothorax in two patients (2/20, 10%). There were no instances of residual patency in the surgical group., Conclusions: Transaxillary thoracotomy without tube thoracostomy and with critical pathway methods allows safe and effective ligation of a patent ductus arteriosus with early hospital discharge. This surgical method has similar cost, higher efficacy rate, and applicability in all patients as compared with newer transcatheter coil occlusion techniques for closure of a patent ductus arteriosus.

Background: Little is known about the incidence, indications, and results of surgical repair or replacement of the aortic valve after balloon aortic valvuloplasty (BAV) for congenital aortic stenosis in children. This study was designed to evaluate patterns of failure requiring operation after BAV for congenital aortic stenosis and to review our experience with successful repair, rather than replacement, of selected aortic valves after BAV., Methods: From March 1986 to June 1995, 60 patients with congenital aortic stenosis aged 1 day to 27 years (mean +/- standard deviation, 7.3 +/- 6 years) underwent BAV. Twenty-three patients (38%) required operation a mean of 44 +/- 37 months (range, 1 to 110 months) after BAV, because of severe aortic insufficiency in 13 patients and recurrent or residual aortic stenosis in 10 patients. Severe aortic insufficiency was invariably due to avulsion of a cusp from the annulus, with resulting cusp prolapse and insufficiency. Operative intervention consisted of valve replacement in 14 patients and valve repair in 9 patients. Repair techniques included reattachment of an avulsed cusp to the aortic annulus, relief of commissural fusion, and debridement of thickened cusps., Results: Actuarial freedom from surgical intervention after BAV was 88% +/- 4% at 1 year, 70% +/- 6% at 5 years, and 51% +/- 12% at 9 years. The need for aortic valve operation was unrelated to age at the time of BAV, indication for operation (aortic insufficiency versus aortic stenosis), age of operation, or preoperative gradient. All patients survived aortic valve operation; there was one late death at an average follow-up of 27 +/- 20 months (range, 2 to 61 months) after aortic valve operation. Stenosis was well relieved in all patients undergoing valve replacement. The 9 valve repair patients have been followed for 22 +/- 14 months (range, 1 to 47 months). Echocardiographic follow-up of the valve repair patients revealed a mean residual aortic stenosis peak instantaneous gradient of 32 mm Hg and mild aortic insufficiency or less in all patients., Conclusions: Aortic valve operation is required in 5% to 7% of patients yearly after BAV. The need for operation appears to be unrelated to age at the time of BAV; aortic insufficiency predominates over aortic stenosis as an indication for operative intervention. Valve repair can be applied in some patients after BAV with good intermediate-term results and may delay the need for aortic valve replacement.

Background: Despite theoretical advantages of absorbable suture in the growing vascular anastomosis, there has not been a documented advantage over nonabsorbable suture in preventing late anastomotic stenosis in total anomalous pulmonary venous connection (TAPVC)., Methods: We reviewed our experience from 1982 to 1994 with 65 hospital survivors of total TAPVC repair to examine the influence of suture type on survival and incidence of late pulmonary venous obstruction. From 1982 until 1988, we used continuous nonabsorbable polypropylene suture for the pulmonary venous-left atrial anastomosis in supracardiac, infracardiac, and mixed types of TAPVC: In 1989, we adopted a running absorbable polydioxanone suture technique. Cardiac catheterization and echocardiography were used to evaluate late pulmonary venous obstruction., Results: Late pulmonary venous obstruction occurred in 17% (4/23) of survivors after repair with polypropylene suture compared with 3.2% (1/32) after repair with polydioxanone suture (p < 0.05). There were no instances of late pulmonary venous obstruction in the intracardiac TAPVC group (0/10). All late pulmonary venous obstructions occurred within 16 months after operation. The actuarial 3-year and 5-year freedom from late pulmonary venous obstruction was 100% for intracardiac TAPVC, 96% for the polydioxanone group, and 81% for the polypropylene group. Five patients died late (5/65, 7.7%), 3 in the polypropylene suture group (3/23, 13%) and 2 in the polydioxanone group (2/32, 6%)., Conclusions: Continuous absorbable polydioxanone suture for the repair of TAPVC results in a low incidence of late pulmonary venous obstruction and death and appears to offer advantages over a continuous nonabsorbable suture. A continuous nonabsorbable suture may limit growth of a vascular anastomosis, particularly one involving a "low-pressure" anastomosis such as in the repair of TAPVC:

At higher elevations, alveolar hypoxia increases pulmonary vascular resistance and may limit the cardiac output of individuals without a subpulmonary ventricle. Thus, we reviewed the outcome of definitive palliation for tricuspid atresia and other forms of single ventricle in 60 consecutive Fontan patients living at a mean elevation of 1,370 m (range, 910 to 2,130 m). There were four early deaths (6.7%; 70% confidence limits, 4.1% to 10.7%) and six late deaths. Kaplan-Meier actuarial survival (+/- standard error) is 79.6% +/- 8.2% at 5 years. Survival was significantly decreased in patients with a preoperative mean pulmonary arterial pressure greater than or equal to 15 mm Hg unless the Fontan procedure was performed with a residual fenestration. Exercise tolerance was significantly worse at higher elevations in a subgroup of patients who have traveled to altitudes of 1,680 to 3,350 m. We conclude that the early and intermediate results of the Fontan procedure at moderately high altitude are similar to results reported at sea level. However, exercise tolerance may be impaired at higher elevations in many patients.

Despite the increasing use of the bidirectional cavopulmonary shunt, little is known about the late results, the duration of palliation, and the frequency with which this procedure allows later successful conversion to a Fontan type of procedure. We reviewed our experience (1984 to 1992) in 38 consecutive children, ages 4 months to 16 years (mean, 4.0 years), who underwent a bidirectional cavopulmonary shunt procedure. All had a single functional ventricle and represented high risks for the performance of a Fontan procedure based on anatomic and hemodynamic criteria. The oxygen saturation in these patients improved from a preoperative value of 75% +/- 7% to 82% +/- 7% (p < 0.05) at late cardiac catheterization by a mean of 24 months after operation. The actuarial survival, including early deaths and that associated with all secondary procedures, was 86% at 1 year and 81% at 6 years. Early deaths occurred in 5.3% (2/38) and late deaths in 11% (4/36). Late follow-up ranged from 5 to 90 months (mean, 37 months). Conversion to a Fontan or fenestrated Fontan procedure was accomplished in 21 early survivors (21/36; 58%) by a mean of 26 months after the bidirectional cavopulmonary shunt procedure, with one operative and no late deaths (1/21; 4.8%). Three additional patients have undergone late reoperation, including 2 requiring cardiac transplantation and 1 undergoing the late creation of an axillary artery-to-vein fistula for the treatment of cyanosis. The midterm survival after a bidirectional cavopulmonary shunt procedure appears to be excellent, and it serves as a good staging procedure for patients who represent high risks for a Fontan procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: The use of balloon coarctation angioplasty instead of surgery as treatment for unoperated coarctation of the aorta is controversial. The efficacy and complications of the two procedures have not been studied before in a prospective fashion., Methods and Results: Thirty-six patients were prospectively randomized to either angioplasty (20 patients) or surgery (16 patients). Immediate results and patient follow-up, including physical examination, angiograms, and magnetic resonance imaging, were compared between groups. Reduction in peak systolic pressure gradient across the coarctation was similar (86%) immediately after both balloon coarctation angioplasty and surgery. On follow-up, aneurysms were seen only in the angioplasty group (20%) and not in the surgery group (0%). No aneurysms have shown progression or required surgery. The incidence of other complications was similar in both groups, although two patients experienced neurological complications after surgery. Although not statistically different, the incidence of restenosis (peak systolic pressure gradient > or = 20 mm Hg) tended to be greater in the angioplasty group (25%) than in the surgery group (6%). Restenosis after angioplasty occurred more frequently in patients with an aortic isthmus/descending aorta diameter ratio < 0.65 and was associated with an immediate catheterization residual peak systolic pressure gradient across the coarctation > or = 12 mm Hg., Conclusions: Immediate gradient reduction is similar after balloon coarctation angioplasty and surgical treatment of unoperated coarctation of the aorta. The risks of aneurysm formation and possibly restenosis after angioplasty are higher than after surgery, although the risks of other complications are similar. Balloon coarctation angioplasty may provide an effective initial alternative to surgical repair of unoperated coarctation of the aorta in children beyond infancy, particularly in patients with a well-developed isthmus. Further follow-up is necessary to determine the long-term risks of postangioplasty aneurysms.

Cholelithiasis has been reported to occur rarely in infants. To determine the incidence of cholelithiasis in infants receiving furosemide, we prospectively performed ultrasonograms on 86 patients. We studied 42 patients receiving furosemide (subjects) and 44 patients not receiving furosemide (controls). There was a significantly higher incidence of gallstones in subjects (21%) than in controls (2%) (P less than .05). When followed over 1 year, the gallstones did not resolve. There were no significant differences in the dosage of furosemide, gestational age, placement of umbilical venous catheters, or amount of total parenteral nutrition (TPN) between subjects with and without gallstones. However, subjects received more days of TPN (16.7 +/- 15.1) than controls (8.4 +/- 13.2) (P less than .05). These data show that the incidence of cholelithiasis is higher than previously suspected in infants receiving furosemide. Thus, furosemide, either independently or in conjunction with the use of TPN, predisposes infants to the development of cholelithiasis.

Activation and deposition of C3 on Aspergillus fumigatus conidia have been previously demonstrated. This study investigated in further detail the interactions between complement component C3 and the conidia of A. fumigatus. Immunoblotting and 125I-C3 binding studies showed that C3 deposition was rapid (less than 15 min) and parallel to the formation of iC3b. Immunoblotting experiments identified a 54- to 58-kDa conidial protein which binds human complement component C3 and/or a C3 fragment(s). 125I labeling of the outer layer of the conidia demonstrated that this protein doublet was present on the surface of the spore. The further degradation of C3 to low-molecular-mass fragments (40, 37, and 30 kDa), in the absence of plasma, by intact living conidia and a preparation of the outer conidial wall layer indicated the ability of fungal components to cleave C3. These data suggest that interactions between conidia and C3 are not limited only to deposition via activation of the alternative complement pathway; they also include degradation of bound C3.

We performed transventricular pulmonary valvotomy as initial surgery in 22 consecutive patients with pulmonary atresia and intact ventricular septum who had a patent infundibulum. Nineteen patients also had placement of a central aortopulmonary shunt. All patients survived surgery, and 16 patients have had preoperative and later postoperative catheterizations. The purpose of this study was to determine the response of the right ventricle to transventricular pulmonary valvotomy with regard to relief of right ventricular hypertension and growth of the entire right ventricle, including tricuspid valve, right ventricular volume, and right ventricular outflow tract. Right ventricular systolic pressure decreased from 111.3 +/- 31.7 mm Hg before initial surgery to 65.6 +/- 26.2 mm Hg. Right ventricular end-diastolic volume increased from 59.1 +/- 39.3% of predicted normal before initial surgery to 114.6 +/- 63.2% at late follow-up catheterization. Tricuspid valve anulus circumference also increased in size from 73.2 +/- 21.3% of predicted normal before initial surgery to 90.4 +/- 22.8% at late follow-up catheterization. Only one patient (6%) required a transanular right ventricular outflow tract patch at the time of biventricular repair. Twenty of 22 patients (91%) either have had or are awaiting biventricular repair. We conclude that transventricular pulmonary valvotomy and central aortopulmonary shunt can be performed safely in newborn infants with pulmonary atresia and intact ventricular septum who have a patent infundibulum. Effective valvotomy relieves right ventricular hypertension, allows for excellent right ventricular and tricuspid valve growth, and optimizes potential for biventricular repair.

From 1986 to 1988, balloon aortic valvuloplasty was performed in 32 patients with congenital valvular aortic stenosis. The patients ranged in age from 2 days to 28 years (mean +/- SD 8.3 +/- 5.9). One balloon was used in 17 patients and two balloons were used in 15 patients. Immediately after valvuloplasty, peak systolic pressure gradient across the aortic valve decreased significantly from 77 +/- 27 to 23 +/- 16 mm Hg (p less than 0.01), a 70% reduction in gradient. At early follow-up study (4.1 +/- 3.3 months after valvuloplasty), there was a 48 +/- 20.5% reduction in gradient compared with that before valvuloplasty, and at late follow-up evaluation (19.2 +/- 5.6 months), a reduction in gradient of 40 +/- 29% persisted. Echocardiography showed evidence of significantly increased aortic regurgitation in 10 patients (31%) and aortic valve prolapse in 7 patients (22%). There was no correlation between the balloon/anulus ratio and the subsequent development of aortic regurgitation or prolapse. In fact, no patient who showed a significant increase in aortic regurgitation had had a balloon/anulus ratio greater than 100%. It is concluded that balloon aortic valvuloplasty effectively reduces peak systolic pressure gradient across the aortic valve in patients with congenital aortic stenosis. However, subsequent aortic regurgitation and prolapse occur in a significant number of patients, even if appropriate technique and a balloon size no greater than that of the aortic anulus are used.

We have reinvestigated whether surface immunoglobulin (sIg) on Trypanosoma lewisi is antibody directed toward parasite antigen by using flow cytometry to analyze parasites stained with fluoresceinated F(ab')2 fragments of antibodies to rat IgG and IgM. We have confirmed that IgG antibody to the parasites is present both in the serum of rats and on the surface of parasites between the fourth and twentieth days of infection, that the amount of sIg per cell increases as the infection progresses, and that considerably more IgG is present on parasites harvested from intact rats than on those from rats that had been immunosuppressed by whole body gamma-irradiation. In addition sIgM was detected on trypanosomes from intact, but not on parasites from irradiated rats. We have also made two observations suggesting that not all sIg is specific antibody made in response to T. lewisi. First, a low but significant amount of sIgG was detected on parasites throughout infection in irradiated rats; no sIgM was detected on these parasite. Second, when parasites harvested from immunosuppressed rats were incubated in normal rat serum, the amount of both sIgG and sIgM detected by flow immunofluorescence increased. Parasites harvested from intact animals bound IgM but not IgG from normal rat serum. These results suggest either that natural antibody to the trypanosomes is present in the serum of uninfected rats or that some rat immunoglobulins bind to structures on the trypanosome surface in ways that do not depend on usual antigen-antibody interactions. Finally, flow immunofluorescence was also used to detect complement component C3 on the surface of both intact and trypsinized bloodstream forms harvested from intact or immunosuppressed rats. The amount of sC3 per cell did not increase until late in the infection and consequently did not correlate with the increase of sIgG. Therefore, T. lewisi avoids destruction by the immune system although immune effector molecules, IgG, IgM, and C3, are on its surface.

Recent work has shown that normal mouse serum (NMS) and plasma contain inhibitory substance(s), termed NMS-In, that suppress proliferative and polyclonal antibody responses elicited in vitro with various B cell mitogens. In this study, the NZB mouse, which has a high degree of spontaneous polyclonal B cell activity, was examined for possible reduced amounts of NMS-In. Also, the capacity of NZB B cells to be inhibited by NMS-In was determined. It was found that sera from NZB mice had normal amounts of NMS-In, as assessed by inhibition of mitogenesis of spleen cells in culture induced by bacterial endotoxin (ET). However, B cells of the NZB mouse were found to be abnormal, requiring approximately 4 to 7 times more NMS-In to inhibit polyclonal antibody synthesis and mitogenesis elicited by ET than B cells of more immunologically normal mice. Removal of T cells by treatment with anti-thy 1.2 and complement did not change the sensitivity of the NZB B cells to NMS-In. Insensitivity to NMS-In was expressed in B cells from relatively young NZB mice, e.g., 9 to 15 wk of age. The relative resistance of B cells of the NZB mouse to regulation by a natural inhibitor in NMS could partially serve to explain their high level of spontaneous antibody synthesis. Furthermore, the fact that B cells of the NZB mouse were ultimately susceptible to inhibition by relatively large amounts of NMS-In affords the opportunity in the future to suppress polyclonal antibody activity in the NZB mouse with purified NMS-In in an attempt to ameliorate autoimmune disease.

Normal mouse serum (NMS) in relatively small amounts was demonstrated to inhibit in vitro splenic B cell activation by several B cell mitogens, polyclonal activators, and T-independent antigens. Proliferative and polyclonal responses elicited by bacterial endotoxin (ET), PPD, or Fc fragment of Ig were suppressed routinely 90% or greater when 0.5% homologous NMS was present in the culture medium. Similar suppression of T-independent antibody responses to TNP-ET and TNP-Ficoll was also observed. In contrast, activation by the T cell mitogen, Con A, and a T-dependent antigen response to SRBC was not inhibited by NMS. Suppression of B cell responses by NMS was not dependent on T cells. Preliminary characterization of NMS-In revealed a m.w. of approximately 200,000, an isoelectric point of 5, and solubility in 40% saturated ammonium sulfate. Both functionally and physically, NMS-In appears to be different from other currently relatively well characterized regulatory substances in serum. NMS-In was found in plasma as well as in serum, suggesting that NMS-In may function naturally in vivo to minimize nonspecific activation of B cells.

The potential for right ventricular growth and physiological repair in tricuspid atresia may influence the type of Fontan procedure. To evaluate preoperative right ventricular assessment, we compared the right ventricular size and morphology determined by selective right ventricular catheterization with axial left ventricular angiography. In seven consecutive patients with tricuspid atresia and ventriculo-arterial concordance, the right ventricular volume was 12.8 +/- 9.4 cc, with a predicted normal volume (based on body surface area) of 31 +/- 16 cc, 43% (range 24-78%) of normal. Right ventricular injection outlined a right ventricular area twice that visualized from an axial left ventricular injection (33.2 vs. 16.5 cm). All patients had a well developed but small trabecular portion of the right ventricle, often unopacified with left ventricular injection. Subinfundibular narrowing adjacent to the ventricular septal defect was invariably present, creating, in effect, a two-chambered right ventricle. Selective right ventriculography demonstrates the unique morphology of the right ventricle in patients with tricuspid atresia not visualized by axial left ventriculography.

The rat parasite Trypanosoma lewisi was incubated in vitro with rat or human serum, washed, and extracted in detergent. Extracts were fractionated by electrophoresis in denaturing gels, transferred to nitrocellulose, allowed to renature, then immunoblotted with polyclonal antibodies to rat complement component C3 and human complement components C3, C5, and factor B. Molecules that reacted with these antibodies were detected in the extracts. Fragments of rat C3 were detected in extracts of parasites that had not been exposed to serum in vitro. Additional complement deposition occurred during in vitro incubations; human complement components deposited in vitro could be distinguished from rat components deposited in vivo. Complement deposition in vitro required magnesium ions and did not occur when heat inactivated serum was used. Components reacting with antibodies to human C3 included a group of bands with molecular weights higher than C3 alpha or beta chains. Blotting with affinity purified, chain specific antibodies demonstrated that a 68 kDa component on parasites is C3 beta and that a 44 kDa molecule is derived from C3 alpha. A 73 kDa component that was difficult to resolve from C3 beta is probably also a C3 alpha fragment. This suggests that an inactive iC3b-like molecule is present on parasites. Kinetic studies showed that cleavage of C3 alpha is rapid and that the amount of C3 alpha fragments and C3 beta on intact parasites reached a steady state after 15 min. When parasites were trypsinized prior to incubation in C5 or C6 deficient serum, the rate and extent of C3 and C5 deposition increased. Unprocessed C3 alpha' and C5 alpha' chains were detected. Trypsinized parasites were lysed by the alternative complement pathway in normal serum. Intact parasites could be lysed by complement in the presence of antibody. The data support our previous suggestion that trypsin sensitive surface proteins on intact T. lewisi limit alternative pathway activity by restricting C3/C5 convertase activity.

Detergent extracts of whole Trypanosoma lewisi were fractionated by polyacrylamide gel electrophoresis and transferred to nitrocellulose for immunoblotting analysis. Antibody probes to rat immunoglobulin G (IgG) and IgM detected intact gamma chains, mu chains, and light chains in extracts. The amount of immunoglobulin detected increased as the infection progressed. Transfers were also incubated in serum from conventionally reared (CRS), specific-pathogen-free (SPFS), or germ-free rats before being probed with anti-rat IgG or anti-rat IgM. Components of 200, 175, and 120 kilodaltons (kDa) bound IgM from all sera tested and were present in extracts of trypanosomes isolated from lethally irradiated or intact rats on different days during infection. No parasite components bound IgG from serum of germ-free rats. However, 145-, 175-, and 200-kDa components bound IgG from CRS and SPFS. A 74-kDa protein was the major IgG-binding component in extracts of reproducing parasites. This component bound much more IgG from CRS than it bound from SPFS. The 74-kDa protein was removed from parasites by mild trypsinization and corresponded to a major surface glycoprotein detected when intact cells were radioiodinated. These results indicate that natural antibodies to T. lewisi exist in rats or that these parasites have surface proteins that bind immunoglobulins without regard to antigenic specificity.

Reproducing forms of Trypanosoma lewisi isolated from X-irradiated rats and adult forms from intact rats were not lysed by fresh mammalian sera. Treating parasites with trypsin or chymotrypsin, but not with neuraminidase, under conditions which did not impair viability rendered the parasites sensitive to lysis by rat, mouse, rabbit, and human sera. Serum from animal strains or humans genetically deficient in complement component C3, C5, or C6 did not lyse protease-treated parasites. The lytic factors in serum displayed the heat sensitivity and the Mg2+ requirement characteristic of the alternate complement pathway. Lysis was resolved into two phases, Mg2+-dependent binding of serum factors to parasites and subsequent C5-dependent, Mg2+-independent lysis. Allowing protease-treated parasites to readsorb host proteins did not block lysis by serum. Protease-treated parasites regenerated components which prevented complement-mediated lysis during 2 h in culture at 37 degrees C. This regeneration was inhibited by cycloheximide but not by tunicamycin. Ten major components were resolved in radioautographs of sodium dodecyl sulfate-polyacrylamide gels of extracts of radioiodinated intact cells. Protease treatment before radioiodination reduced the amount of radioactivity associated with these components disproportionately. Components with apparent molecular weights of 102,000, 88,000, and 47,000 were strongly labeled in intact cells, poorly labeled after enzyme treatment, and again labeled in cells that were cultured at 37 degrees C after enzyme treatment. Cycloheximide blocked the reappearance of these components on cultured cells. The presence of these three components was therefore correlated with resistance to complement-mediated lysis.