Fatal Scopulariopsis infection in a lung transplant recipient: lessons of organ procurement.

Seventeen days after double lung transplantation, a 56-year-old patient with idiopathic pulmonary fibrosis developed respiratory distress. Imaging revealed bilateral pulmonary infiltrates with pleural effusions and physical examination demonstrated sternal instability. Broad-spectrum antibacterial and antifungal therapy was initiated and bilateral thoracotomy tubes were placed. Both right and left pleural cultures grew a mold subsequently identified as Scopulariopsis brumptii. The patient underwent pleural irrigation and sternal debridement three times but pleural and wound cultures continued to grow S. brumptii. Despite treatment with five antifungal agents, the patient succumbed to his illness 67 days after transplantation. Autopsy confirmed the presence of markedly invasive fungal disease and pleural rind formation. The patient's organ donor had received bilateral thoracostomy tubes during resuscitation in a wilderness location. There were no visible pleural abnormalities at the time of transplantation. However, the patient's clinical course and the location of the infection, in addition to the lack of similar infection in other organ recipients, strongly suggest that Scopulariopsis was introduced into the pleural space during prehospital placement of thoracostomy tubes. This case of lethal infection transmitted through transplantation highlights the unique risk of using organs from donors who are resuscitated in an outdoor location.

Relationship between patient complaints and surgical complications.

BACKGROUND: Patient complaints are associated with increased malpractice risk but it is unclear if complaints might be associated with medical complications. The purpose of this study was to determine whether an association exists between patient complaints and surgical complications. METHODS: A retrospective analysis of 16,713 surgical admissions was conducted over a 54 month period at a single academic medical center. Surgical complications were identified using administrative data. The primary outcome measure was unsolicited patient complaints. RESULTS: During the study period 0.9% of surgical admissions were associated with a patient complaint. 19% of admissions associated with a patient complaint included a postoperative complication compared with 12.5% of admissions without a patient complaint (p = 0.01). After adjusting for surgical specialty, co-morbid illnesses and length of stay, admissions with complications had an odds ratio of 1.74 (95% confidence interval 1.01 to 2.98) of being associated with a complaint compared with admissions without complications. CONCLUSIONS: Admissions with surgical complications are more likely to be associated with a complaint than surgical admissions without complications. Further research is necessary to determine if patient complaints might serve as markers for poor clinical outcomes.

Real-time ultrasound measure of the fetal eye (vitreous body) for prediction of parturition date in small ponies.

Ultrasonographic fetal eye measures have been used to estimate gestational age of the fetus in light horse mares. However, fetal eye measures have not been published for smaller pony breeds. This study was conducted to develop reference ranges for ultrasonographic measures of fetal eyes in small ponies for the purpose of predicting days before parturition (DBP) when breeding or ovulation dates are unknown. Twenty-three Shetland-type pony mares were studied across one (n = 10) or two (n = 13) gestations in 2004 (18 pregnancies) and 2005 (18 pregnancies). Measurements of fetal eyes were obtained during transrectal ultrasound examination. Examinations were conducted once monthly in a field situation beginning in December (2003) or August (2004) until mares foaled (March through July). The length (from sclera to sclera) and width (from retina to cornea) of the vitreous body were measured. For the 273 examinations in which gestation age was greater than 2 months, eye measures were obtainable in 248 (91%). Mixed-effects linear regression modeling was used to account for serial growth measures within pregnancy, repeated measurements across mares, and unbalanced study design. Independent variables evaluated included vitreous body length, vitreous body width, the ratio of length to width, parity, and mare height at the withers at parturition. Eye length was the best single predictor of days before parturition, with almost no additional predictive value of the other variables considered. Our resulting regression equation is: days before parturition=265.16-0.21*(vitreous body length in mm)(2). This study suggests that measure of the fetal eye is a practical on-farm procedure for estimating days before parturition in small ponies.

Laparoscopic adrenalectomy: 100 resections with clinical long-term follow-up.

BACKGROUND: The operative results of 100 laparoscopic adrenal resections in 94 patients and the subsequent impact on postoperative antihypertensive therapy are presented. METHODS: Clinical and follow-up data for resections performed between 1995 and 2003 were obtained from medical records, patient questionnaires, and telephone interviews. RESULTS: The diseases included Conn's syndrome (27 patients), Cushing's syndrome (30 patients), pheochromocytoma (11 patients), and Other tumors (26 patients). Antihypertensive therapy was eliminated or reduced for Conn's syndrome (75%), Cushing's syndrome (27%), pheochromocytoma (88%) and patients with Other tumors (54%). Clinical improvement was observed by 12 months for pheochromocytoma patients as compared with 35 to 45 months for the other groups (p < 0.05). Multivariate analysis showed that pheochromocytoma patients were more likely to experience improvement or cure than the Other tumor group (hazard ratio, 4.87; 95% confidence interval, 1.61-14.7). CONCLUSIONS: Laparoscopic adrenalectomy continues to be safe and efficacious for benign adrenal diseases. Although patients with functional tumors can expect improvement or cure, the time until improvement may be longer than previously recognized.

Cost perspectives of laparoscopic and open appendectomy.

BACKGROUND: Despite multiple studies comparing laparoscopic and open appendectomies, the clinically and economically superior procedure still is in question. A cost analysis was performed using both institutional and societal perspectives. METHODS: A decision analytic model was developed to evaluate laparoscopic and open appendectomies. The institutional perspective addressed direct health care costs, whereas the societal perspective addressed direct and indirect health care costs. Baseline values and ranges were taken from randomized controlled trials, meta-analyses, and Medicare databases. RESULTS: From the institutional perspective, open appendectomy is the least expensive strategy, with an expected cost of $5,171, as compared with $6,118 for laparoscopic appendectomy. The laparoscopic approach is less expensive if open appendectomy wound infection rates exceed 23%. From the societal perspective, laparoscopic appendectomy is the least expensive strategy, with an expected cost of $10,400, as compared with $12,055 for open appendectomy. CONCLUSIONS: The decision analysis demonstrated an economic advantage to the hospital of open appendectomy. In contrast, laparoscopic appendectomy represents a better economic choice for the patient.

Kaposi's sarcoma-associated herpesvirus open reading frame 50/Rta protein activates the entire viral lytic cycle in the HH-B2 primary effusion lymphoma cell line.

Rta, the gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. What was not demonstrated in previous studies was whether KSHV Rta was competent to initiate the entire viral lytic life cycle including lytic viral DNA replication, late-gene expression with appropriate kinetics, and virus release. In HH-B2, a newly established primary effusion lymphoma (PEL) cell line, KSHV ORF50 behaved as an immediate-early gene and autostimulated its own expression. Expression of late genes, ORF65, and K8.1 induced by KSHV Rta was eliminated by phosphonoacetic acid, an inhibitor of viral DNA polymerase. Transfection of KSHV Rta increased the production of encapsidated DNase-resistant viral DNA from HH-B2 cells. Thus, introduction of an ORF50 expression plasmid is sufficient to drive the lytic cycle to completion in cultured PEL cells.

Dihydrofolate reductase from Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the first human virus known to encode dihydrofolate reductase (DHFR), an enzyme required for nucleotide and methionine biosynthesis. We have studied the purified KSHV-DHFR enzyme in vitro and analyzed its expression in cultured B-cell lines derived from primary effusion lymphoma (PEL), an AIDS-associated malignancy. The amino acid sequence of KSHV-DHFR is most similar to human DHFR (hDHFR), but the viral enzyme contains an additional 23 amino acids at the carboxyl-terminus. The viral DHFR, overexpressed and purified from E. coli, was catalytically active in vitro. The K(m) of KSHV-DHFR for dihydrofolate (FH(2)) was 2.4 microM, which is significantly higher than the K(m) of recombinant hDHFR (rhDHFR) for FH(2) (390 nM). K(m) values for NADPH were similar for the two enzymes, about 1 microM. KSHV-DHFR was inhibited by folate antagonists such as methotrexate (K(i): 200 pM), aminopterin (K(i): 610 pM), pyrimethamine (K(i): 29 nM), trimethoprim (K(i): 2.3 microM), and piritrexim (K(i): 3.9 nM). In all cases, K(i) values for these folate antagonists were higher for KSHV-DHFR than for rhDHFR. The viral enzyme was expressed at levels two- to tenfold higher than hDHFR in PEL cell lines as an early lytic cycle gene. KSHV-DHFR mRNA and protein appeared from 6 to 24 h after chemical induction of the KSHV lytic cycle. Epitope-tagged KSHV-DHFR and rhDHFR both localized to the nucleus of transfected cells, while other KSHV nucleotide metabolism genes localized to the cytoplasm. DHFR activity was not essential for viral replication in cultured PEL cells. Since hDHFR was not detectable in peripheral blood mononuclear cells (PBMCs), KSHV-DHFR may function to provide increased DHFR activity in vivo in infected cells that have little or none of their own enzyme.

Debeerius ellefseni (Fam. Nov., Gen. Nov., Spec. Nov.), an autodiastylic chondrichthyan from the Mississippian bear gulch limestone of Montana (USA), the relationships of the chondrichthyes, and comments on gnathostome evolution.

Debeerius ellefseni is an autodiastylic, operculate chondrichthyan from the 320-million-year-old Bear Gulch limestone (Heath Formation, Big Snowy Group, Upper Chesterian) of Montana, USA. Cranial and postcranial morphologies show strong affinities to the holocephalan cochliodonts and Chimaeriformes. The heterodont dentition is, however, selachian in plan. Debeerius ellefseni's cranial, postcranial, and suspensorial characters identify this fish as a paraselachian, an early chondrichthyan with a morphology intermediate to the chimaeroid and selachian plans. They also support the division of Chondrichthyes into the subclasses Elasmobranchii and Euchondrocephali (Paraselachii + Holocephalimorpha). Details of the anatomy of D. ellefseni are reviewed in light of recent advances in understanding vertebrate splanchnocranial development and, thus, permit a discussion of historically problematic craniate features, including labial cartilages and the nature of the mandibular arch relative to hyoid and branchial arches. Developmental and evolutionary considerations of these characters are consistent with an embryonic body plan shared by both lampreys and gnathostomes. Debeerius ellefseni's suspensorium corresponds to the plesiomorphous gnathostome condition theorized by DeBeer and Moy-Thomas in 1935. The description of this autodiastylic condition is clarified to include observations of the hyoid arch, which is complete with a pharyngohyal and provides support for the primary opercular valve. The confirmation of an autodiastylic suspensorium requires a reexamination of the commonly accepted paradigm for jaw evolution. The selachian, chimaeroid, and actinopterygian conditions are all derivable from this plesiomorphous state; the placoderm and sarcopterygian conditions are related and probably similarly derived. The comparable osteichthyan suspensorium is best represented by the suspensorial condition of coelacanths.

Kinetics of Kaposi's sarcoma-associated herpesvirus gene expression.

Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the "immediate-early" gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.

Management of incidentally discovered cervical paragangliomas: report of two cases and review of current issues.

Paragangliomas of the head and neck are uncommon neoplasms arising from the extra-adrenal paraganglia and include carotid body and glomus vagale tumors. These lesions may be discovered incidentally by imaging studies performed to evaluate carotid atherosclerotic occlusive disease. Incidental paragangliomas of the head and neck may be smaller than those discovered due to symptoms. Although surgical resection remains the definitive treatment for head and neck paragangliomas, important issues of management arise when such lesions are discovered. Two recent cases are reported. Epidemiology, pathophysiology, diagnostic evaluation, and issues of management of head and neck paragangliomas are discussed.

Selective switch between latency and lytic replication of Kaposi's sarcoma herpesvirus and Epstein-Barr virus in dually infected body cavity lymphoma cells.

The BC-1 cell line, derived from a body cavity-based, B-cell lymphoma, is dually infected with Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In these studies, the relationships between these two gammaherpesviruses and BC-1 cells were characterized and compared. Single-cell cloning experiments suggested that all BC-1 cells contain both genomes. In more than 98% of cells, both viruses were latent. The two viruses could be differentially induced into their lytic cycles by chemicals. EBV was activated into DNA replication and late-gene expression by the phorbol ester tetradecanoyl phorbol acetate (TPA). KSHV was induced into DNA replication and late-gene expression by n-butyrate. Amplification of both EBV and KSHV DNAs was inhibited by phosphonoacetic acid. Induction of the KSHV lytic cycle by n-butyrate was accompanied by the disappearance of host-cell beta-actin mRNA. Induction of EBV by TPA was not accompanied by such an effect on host-cell gene expression. Induction of the KSHV lytic cycle by n-butyrate was associated with the expression of several novel polypeptides. Recognition of one of these, p40, served as the basis of development of an assay for antibodies to KSHV in the sera of infected patients. BC-1 cells released infectious EBV; however, there was no evidence for the release of encapsidated KSHV genomes by BC-1 cells, even though n-butyrate-treated cells contained numerous intranuclear nucleocapsids. The differential inducibility of these two herpesviruses in the same cell line points to the importance of viral factors in the switch from latency to lytic cycle.

N- and C-terminal substance P fragments modulate striatal dopamine outflow through a cholinergic link mediated by muscarinic receptors.

The present study investigated whether the modulatory effects of substance P and substance P fragments on striatal dopamine release involve a cholinergic link. Rat striatal slices were incubated with substance P, substance P(1-4), substance P(1-7), substance P(5-11) and substance P(8-11) in the absence or presence of various agents which modify cholinergic transmissions, and endogenous dopamine outflow was measured using high-performance liquid chromatography. The incubation of striatal slices with substance P and its N- and C-terminal fragments (1 nM) induced a significant overflow of endogenous dopamine. Neostigmine (150 nM) potentiated the effects of substance P and its fragments, whereas the incubation with hemicholinium-3 (50 microM) abolished the effects of the peptides on dopamine outflow. The acetylcholinesterase inhibitor and the inhibitor of choline uptake did not have intrinsic effects on dopamine outflow. The muscarinic antagonist atropine (1 microM) reversed completely the effects of substance P and its fragments, whereas the nicotinic antagonists dihydro-beta-erythroidine (0.5 microM) and pempidine (10 microM) were devoid of effects. None of the cholinergic antagonists modified dopamine outflow. The results suggest that substance P and several N- and C-terminal substance P fragments activate cholinergic neurons in striatal slices. The released acetylcholine induces an increased dopamine outflow, mediated by muscarinic receptors. These observations represent additional evidence which supports the functional interactions between substance P, acetylcholine and dopamine in the striatum. Furthermore, they show that substance P fragments may exert neuromodulatory effects through mechanisms similar to those underlying the effects of the parent peptide.

Analytical performance of an immunologic-based periodontal bacterial test for simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia.

The analytical performance of a membrane-based immunoassay for the simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia (including Prevotella nigrescens) was investigated. Positive reactions were observed for 71 of 71 reference strains and recent oral isolates of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. No cross-reactivity was observed with 39 other common oral and environmental species. The specificity of the test was unaffected by the presence of potential oral interferents including whole blood, white blood cells, mucin, saliva, toothpastes, and oral rinses. A proficiency test by dental professionals using a standardized set of unknown simulated samples yielded a sensitivity of 97% (116/120) and a 100% specificity (240/ 240). An additional group including dental professionals and high school students was shown to be 99% proficient (1385/1397) in distinguishing proper from improper test function when processing control samples with normal test devices and devices with simulated error conditions. Comparisons to a culture standard for 104 subgingival plaque samples collected from 26 adult periodontitis patients yielded > 98% specificity for each of the test bacteria. In addition, the detection threshold for the test was determined to be equivalent to 10(4) cultivable test bacteria when compared to the culture standard. The data indicate that this membrane immunoassay is a valid and easy-to-use method for the detection of A. actinomycetemcomitans, P. gingivalis, and P. intermedia in subgingival plaque, at levels above the detection threshold of the test.

Antibodies to butyrate-inducible antigens of Kaposi's sarcoma-associated herpesvirus in patients with HIV-1 infection.

BACKGROUND: The recent identification in patients with Kaposi's sarcoma of DNA sequences with homology to gammaherpesviruses has led to the hypothesis that a newly identified virus, Kaposi's sarcoma-associated herpeslike virus (KSHV), has a role in the pathogenesis of Kaposi's sarcoma. We developed serologic markers for KSHV infection. METHODS: KSHV antigens were prepared from a cell line (BC-1) that contains the genomes of both KSHV and the Epstein-Barr virus (EBV). We used immunoblot and immunofluorescence assays to examine serum samples from 102 patients with human immunodeficiency virus type 1 (HIV-1) infection for antibodies to KSHV-associated proteins and to distinguish these antibodies from antibodies to EBV antigens. A positive serologic response was defined by the recognition of an antigenic polypeptide, p40, in n-butyrate-treated BC-1 cells and by the absence of p40 recognition in untreated BC-1 cells or EBV-infected, KSHV-negative cells. The detection by the immunofluorescence assay of 10 to 20 times more antigen-positive cells in n-butyrate-treated BC-1 cells than in untreated cells was considered a positive response. RESULTS: Antibodies to the p40 antigen expressed by chemically treated BC-1 cells were identified in 32 of 48 HIV-1-infected patients with Kaposi's sarcoma (67 percent), as compared with only 7 of 54 HIV-1-infected patients without Kaposi's sarcoma (13 percent). These results were confirmed by an immunofluorescence assay. The positive predictive value of the serologic tests for Kaposi's sarcoma was 82 percent, and the negative predictive value 75 percent. CONCLUSIONS: The presence of antibodies to a KSHV antigenic peptide correlates with the presence of Kaposi's sarcoma in a high-risk population and provides further evidence of an etiologic role for KSHV.

Transmissible retrovirus in Epstein-Barr virus-producer B95-8 cells.

Epstein-Barr virus (EBV) released from the B95-8 marmoset cell line has served as a prototype for biologic and biochemical studies of EBV. Here we identify and characterize a retrovirus carried by many cultures of B95-8 cells. The experiments were stimulated by the isolation of a cDNA clone from B95-8 cells in which sequences from the EBV large internal repeat were linked to gag sequences similar to those of squirrel monkey retrovirus, human isolate, SMRV-H. However, among 413 amino acids predicted from the nucleotide sequence of the gag region of the B95-8 SMRV isolate there were 48 amino acid changes that distinguished this virus from SMRV-H originally isolated from a human lymphoid cell line by Oda et al. (1988, Virology 167, 468-476). Nucleic acid and antibody probes were developed for the B95-8 isolate of SMRV. Using such probes, we found that SMRV-B95-8 was readily transmissible, independent of EBV, as an infectious virus to human B and T cell lines. SMRV-B95-8 was highly fusogenic in the presence or absence of EBV. The ultrastructural appearance of the B95-8 retrovirus was characteristic of a type D retrovirus. Cells dually infected with EBV and SMRV-B95-8 did not demonstrate increased levels of lytic EB viral replication. SMRV-B95-8 did not by itself cause lymphocyte immortalization or enhance immortalization by EBV. Thus SMRV-B95-8 does not contribute to the major biologic properties of the B95-8 strain of EBV.

Hemodynamic and volumetric response of the normal left ventricle to upright tilt testing.

The purpose of this study were to determine the specificity of the head-up tilt test in normal subjects when a graded isoproterenol infusion is used, and to evaluate the role of dynamic ventricular volume change during head-up tilt as a mechanism of syncope. We prospectively studied 12 normal volunteers, each of whom underwent an upright tilt test for 10 minutes at 80 degrees with and without an infusion of isoproterenol. A subgroup of five subjects had a third tilt test during administration of a combination of esmolol and isoproterenol. Blood pressure, heart rate, and left ventricular volumes and flow (obtained with Doppler echocardiography) were recorded in the following sequence: while supine, during upright tilt, while supine with isoproterenol, and during upright tilt with isoproterenol. During the initial head-up tilt, one subject had syncope. An additional eight subjects had presyncope or syncope during head-up tilt with isoproterenol. The remaining three subjects were asymptomatic. In subjects with syncope or near-syncope ("responders"), heart rate increased with isoproterenol but decreased markedly, to 76 +/- 5 beats/min, by the end of the protocol. Systolic blood pressure rose slightly above baseline during isoproterenol but fell from 118 +/- 4 to 85 +/- 5 mm Hg during head-up tilt with isoproterenol. The three asymptomatic subjects had only one significant change, an increase in heart rate with isoproterenol. In the five responders undergoing three tilt tests, left ventricular volume decreased significantly at end diastole (94 +/- 25 vs 58 +/- 22 ml) and end systole (34 +/- 13 vs 18 +/- 6 ml) when supine baseline is compared with initial upright tilt.(ABSTRACT TRUNCATED AT 250 WORDS)

Changing Epstein-Barr viral ZEBRA protein into a more powerful activator enhances its capacity to disrupt latency.

The Epstein-Barr viral transcriptional activator ZEBRA induces expression of viral early lytic genes when introduced into cells bearing latent Epstein-Barr virus. We show here that a ZEBRA-herpes simplex viral protein 16 (VP16) fusion protein induces early viral lytic gene expression in Epstein-Barr virus-containing cells more efficiently than does wild-type ZEBRA. The fusion protein is also a more powerful transcriptional activator in these cells, as assayed with reporter constructs. Our experiments also suggest that ZEBRA manifests a function required for full activity on certain natural promoters but not on promoters bearing oligomerized ZEBRA binding sites; this function cannot be provided by VP16.