BACKGROUND: Inhibition of hyperacute rejection (HAR) and sustained graft survival have been demonstrated in a pig-to-primate model of heterotopic cardiac xenotransplantation using pigs transgenic for human Decay Accelerating Factor (hDAF). Building on this work, an orthotopic model has been developed. This case records 39-day cardiac xenograft function in a life-supporting capacity with clinically applicable immunosuppression. METHODS: Using a heart from an hDAF transgenic pig, an orthotopic cardiac transplant was performed on an adult baboon. The immunosuppressive regimen consisted of induction with a short course of cyclophosphamide, followed by maintenance therapy with cyclosporine A, mycophenolate mofetil and a tapering course of corticosteroids. Post-operative monitoring included daily anti-pig hemolytic antibody titer surveillance and endomyocardial biopsy. RESULTS: The animal survived 39 days and was active and energetic throughout its postoperative course, remaining free of signs of cardiopulmonary failure. Endomyocardial biopsy performed on post-operative Day 36 revealed only patches of sub-endocardial fibrosis with no signs of active rejection. The baboon succumbed to an acute cardiopulmonary decompensation immediately following administration of medication via oral gavage. Post-mortem histopathology demonstrated well-preserved myocardial architecture with small foci of mild humoral rejection. CONCLUSIONS: This case documents the longest survival recorded to date of a discordant orthotopic cardiac xenograft and illustrates that the hDAF transgene combined with a clinically acceptable maintenance immunosuppressive regimen enables sustained, life-supporting function of porcine cardiac xenografts in non-human primates. The inhibition of hyperacute rejection and the subsequent control of humoral and cellular rejection for over 1 month demonstrated in this experiment represent significant progress in the development of a viable strategy for clinical xenotransplantation.

Animals↗

PubMed2000

Growth of porcine kidneys in their native and xenograft environment.

The increased survival of hDAF pig-to-primate renal xenografts for up to two months has afforded the opportunity to study physiological aspects such as organ growth. Experimental evidence exists of species restriction of the activity of growth hormone, although growth itself is also controlled by a number of other endocrine, paracrine and autocrine substances. This study consisted of four parts: (1) measurement of pig kidney size according to pig body weight; (2) measurement of pig kidney size according to pig age; (3) serial length measurement of pig-to-primate renal xenografts; (4) correlation of terminal weight of renal xenograft with age and histology. The xenografted pig kidneys in a primate recipient grow as they would in the pig for the first two weeks after transplantation. After this time there is a reduction in the rate of increase in the length of the xenograft. Over the same period, changes in weight are greatly increased by the presence of rejection. This observational study supports the notion that regulation of growth of a xenotransplanted porcine kidney occurs.

Animals↗

PubMed2000

Acute vascular rejection is associated with systemic complement activation in a pig-to-primate kidney xenograft model.

The introduction of h-DAF transgenic porcine organs into pre-clinical pig-to-primate discordant xenotransplantation has led to complete and reliable abrogation of hyperacute xenograft rejection (HAR). Despite additional heavy immunosuppression however, most xenografts are still lost due to acute vascular rejection (AVR), with current treatment protocols being of only limited value. In a life-supporting model of pig-to-primate kidney transplantation, unmodified (n=8) or h-DAF-transgenic (n=9) porcine kidneys were transplanted into cynomolgus monkeys under cyclophosphamide (CyP), cyclosporine and low-dose steroid immunosuppression. Longest recipient survival was 11 days in the control group and 68 days in the h-DAF transgenic group. Stable initial graft function with recipient survival >4 days was generated in eight animals (two controls and six transgenics). In these animals, plasma complement levels were analyzed during ongoing AVR. Compared with baseline levels, a two-fold increase in C3a levels and a four-fold increase in sC5b-9 levels were measured. In parallel to systemic complement activation, increased deposition of C3 and C5b-9 along with massive staining for recipient IgM immunoglobulins was detected in the xenografts on immunohistochemistry. We conclude that acute vascular xenograft rejection of porcine kidneys in cynomolgus monkeys is associated with classical pathway complement activation following binding of induced recipient anti-porcine antibodies. This complement activation can be observed despite membrane bound expression of human complement regulators in the porcine xenografts. Therefore, additional short-term fluid phase complement inhibition seems necessary for the future development of protocols designed for treatment of AVR in the pig-to-primate combination.

Acute Disease↗

PubMed2000

AglU, a protein required for gliding motility and spore maturation of Myxococcus xanthus, is related to WD-repeat proteins.

The aglU gene of Myxococcus xanthus encodes a protein similar to Het-E1 (vegetative incompatibility) from Podospora anserina, acylaminoacyl-peptidase from Bacillus subtilis, and TolB from Escherichia coli. These proteins all have evenly spaced SPDG repeats that are characteristic of a larger motif called the WD-repeat. The WD-repeat is predicted to form a beta-propeller structure that mediates the assembly of heteromeric protein complexes. AglU has a consensus lipoprotein attachment motif that includes a type II signal sequence followed by a cysteine residue. This suggests that AglU is matured, then attached to the outer membrane via fatty acid acylation at this Cys. Cells carrying a mutation in aglU are blocked in adventurous gliding and can swarm only if cells are in contact with one another. When starved of nutrients, the aglU mutant aggregates and forms multicellular fruiting bodies like the wild-type strain, but is unable to produce heat-resistant spores. This suggests that adventurous gliding motility, per se, is not required for development, but that AglU is essential for a terminal step of spore differentiation.

Amino Acid Sequence↗

PubMed2000

Progressive genetic aberrations detected by comparative genomic hybridization in squamous cell cervical cancer.

Genetic changes orchestrated by human papillomaviruses are the most important known factors in carcinogenesis of the uterine cervix. However, it is clear that additional genetic events are necessary for tumour progression. We have used comparative genomic hybridization to document non-random chromosomal gains and losses within a subset of 37 cervical carcinomas matched for clinical stage Ib, but with different lymph node status. There were significantly more chromosomal changes in the primary tumours when the lymph nodes were positive for metastases. The most frequent copy number alterations were loss of 3p, 11q, 6q and 10q and gain of 3q. The smallest areas of loss and gain on chromosome 3 were 3p14-22 and 3q24-26. The study identifies progressive DNA copy number changes associated with early-stage invasive cervical cancers with and without lymph node metastases, a factor of potential prognostic and therapeutic value.

Adult↗

PubMed2000

Acetone-4-methylthiosemicarbazone at 220 K.

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Anti-Infective Agents↗

PubMed2000

Recent advances in methods for the assessment of dental calculus--research and clinical implications.

The diagnostic evaluation of supra- and subgingival calculus has remained an important research problem for over 40 years. The measurement of calculus remains an important objective in both research and office settings. As might be expected, the most important advances in diagnostic methods for calculus assessment have been derived from cross-fertilization from other technical disciplines including engineering, optics and computer science fields. With the new methods described herein, clinicians and researchers can look forward to important advances in our understanding of the impact of supragingival and subgingival calculus on oral health status and periodontal disease progression and in the development of treatment modalities for improved calculus control, benefiting patients and clinicians alike.

Dental Calculus↗

PubMed2000

Increased cardiomyocyte intracellular calcium during endotoxin-induced cardiac dysfunction in guinea pigs.

Septic shock is a complex pathophysiologic state characterized by circulatory insufficiency, multiple system organ dysfunction, and frequent mortality. Although profound cardiac dysfunction occurs during sepsis, the pathogenesis of this dysfunction remains poorly understood. To determine whether abnormalities in intramyocyte calcium accumulation might contribute to the development of cardiac dysfunction, we measured myocyte intracellular calcium during peak cardiac dysfunction after an endotoxin challenge. Intraperitoneal administration of Escherichia coli lipopolysaccharide 4 mg/kg to adult guinea pigs resulted in significantly impaired cardiac performance (Langendorff preparation) 18 h after challenge compared with control. This included diminished left ventricular pressure development (56 +/- 7 versus 95 +/- 4 mm Hg, p < 0.05), maximal rate of left ventricular pressure rise (998 +/- 171 versus 1784 +/- 94 mm Hg/s, p < 0.05) and left ventricular pressure fall (1014 +/- 189 versus 1621 +/- 138 mm Hg/s, p < 0.05). Assay of intracellular calcium in fura-2AM-loaded cardiac myocytes demonstrated increased intracellular calcium concentration in myocytes obtained from lipopolysaccharide-challenged animals compared with controls (234 +/- 18 versus 151 +/- 6 nM, p < 0.05). Inhibition of calcium-release channel (ryanodine receptor) opening by administration of dantrolene prevented the increase in intracytoplasmic calcium (159 +/- 8 versus 234 +/- 18 nM, p < 0.05) and partially ameliorated systolic and diastolic ventricular dysfunction. These data indicate that abnormalities of intracellular calcium contribute to the development of endotoxin-induced myocardial dysfunction.

Animals↗

PubMed2000

Progress in xenotransplantation.

BACKGROUND: XT using the pig as a donor species may provide a potential solution to the lack of human organs available for transplantation. However, two major immunological obstacles have impeded the survival of porcine organs transplanted into primates. The first is hyperacute rejection (HR), which is a consequence of the recipient's preformed antibodies binding specifically to the carbohydrate structure Gal-alpha1-3-Gal on porcine endothelial cells (EC), leading to complement activation and graft failure. Conventional strategies to overcome HR have focused on the inactivation or removal of essential components of the complement system, or removal of anti-xenograft antibodies from the blood of the recipient. Alternatively, genetically modified donor pigs, whose organs express human complement inhibitors such as human decay-accelerating factor (hDAF), have been produced. AIM: Several groups have shown that organs from these transgenic pigs do not undergo HR when transplanted into primates. Acute vascular rejection (AVR) is the second major immunological obstacle to successful XT. Several strategies are presently being investigated to overcome this form of rejection. The first approach is aimed at controlling the induced anti-xenograft humoral response with immunosuppressive agents primarily directed against lymphocytes. A second approach is aimed at preventing EC activation in the graft by inhibition of NF-kappaB, a transcription factor which is known to play a central role in EC activation. A third approach is aimed at preserving the antithrombotic properties of EC which usually disappear when these cells are activated. Finally, other groups are attempting to tackle AVR by exploring the role of platelet inhibitors or by using antibodies directed against adhesion molecules. To date, the speed and strength of the humoral xenograft rejection process have impeded in vivo studies of the cell-mediated immune response in the pig-to-primate model. However, there is now evidence that human T cells respond vigorously to pig MHC antigens and some authors have proposed that the induction of tolerance is essential if XT is to succeed clinically. It is clear that additional in vivo data still need to be generated in order to fully comprehend the involvement of the cellular immune response in this model. CONCLUSIONS: It has now been demonstrated that organs from hDAF transgenic pigs sustain the life of primates for up to 12 weeks before failing due to the onset of AVR. It is anticipated that once AVR has been overcome, long-term survival of porcine organs transplanted into primates should be reproducibly achievable.

Acute Disease↗

PubMed2000

Effects of tooth-whitening gels on enamel and dentin ultrastructure--a confocal laser scanning microscopy pilot study.

Confocal laser scanning microscopy (CLSM) in reflection mode provides a useful means for nondestructive microscopic examination of the ultrastructural characteristics of hard tissues, including enamel and dentin. In this study, CLSM was used to examine the effects of in vitro bleaching on enamel and dentin. The crowns of extracted human third molars were sectioned below the occlusal fissure, revealing subsurface dentin and the outer surface ring of enamel. Specimens were polished with 1,200-grit alumina followed by fine polish lapidary film, and cut into four equal sections per tooth, allowing each tooth to serve as internal control. Sections were mounted in acrylate for handling and were bleached for 0, 15, and 30 hours in 0.25 g of commercial Opalescence whitening gel (10% carbamide peroxide), the 5.3% hydrogen peroxide gel used in Crest Whitestrips, or 5% HClO4 solution. Blank glycerin served as a control treatment. In vitro whitening was confirmed by colorimeter readings of delta b and delta L with a PR 650 spectrophotometer/colorimeter. Treated teeth were examined with CLSM comparing enamel surface, dentoenamel junction (DEJ), and dentin at approximately 5 microns subsurface to the polished surfaces under an oil immersion objective. A single sample from each treatment (30-hour whitening) was also examined by environmental variable pressure scanning electron microscopy (VP-SEM). Internal comparison of glycerin (no whitening) controls (0-, 15-, and 30-hour exposures) revealed reproducible ultrastructure, facilitating treatment comparisons. Whitened teeth revealed no significant micromorphological changes associated with the whitening process in subsurface enamel, DEJ, and dentin areas. The direct treatment of cross sections permitted simple access of all areas to whitening gel, thereby eliminating the possibility of diffusion limitation of the gel in producing artifacts. VP-SEM observations similarly showed no significant changes in the surfaces after treatment.

Carbamide Peroxide↗

PubMed1999

Poor penetration of the male genital tract by HIV-1 protease inhibitors.

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Drug Therapy, Combination↗

PubMed1999

Calcium antagonists improve cardiac mechanical performance after thermal trauma.

Burn trauma initiates a pathophysiologic cascade, which includes cardiac dysfunction and intramyocyte calcium accumulation. This study examined the hypothesis that therapeutic interventions which limit intracellular cardiac Ca(2+) accumulation after burn trauma will improve cardiac function. Guinea pigs were anesthetized (methoxyflurane), burned over 43% of total body surface area, and fluid resuscitated (FR) for 24 h. Burn guinea pigs were randomly divided into three groups: Group 1, FR alone, Group 2, FR plus dantrolene (10 mg/kg body wt, IV, 30 min, 8 and 22 h postburn), a drug which inhibits the Ca(2+) release channel (ryanodine receptor) of the cardiac sarcoplasmic reticulum, and Group 3, FR plus diltiazem (0.20-0.22 mg/kg given IV as a slow infusion over 6 h postburn), a drug which specifically blocks Ca(2+) slow channels; sham burn guinea pigs were given vehicle (Group 4), dantrolene (Group 5), or diltiazem (Group 6) as described above (respective controls). Cardiac dysfunction was impaired in fluid-treated burns (Group 1) compared to sham burns (Group 4) as indicated by reduced developed left ventricular pressure (LVP) (86 +/- 2 vs 52 +/- 3 mm Hg, P < 0.05), rate of LVP rise, (+dP/dt max, 1379 +/- 64 vs 909 +/- 44 mm Hg/s, P < 0.05), and LVP fall (-dP/dt max, 1184 +/- 31 vs 881 +/- 40 mm Hg/s, P < 0.05), and time to peak pressure (110 +/- 2 vs 102 +/- 2 ms, P < 0.05). In addition, [Ca(2+)](i) rose in cardiomyocytes harvested from fluid-treated burns (Group 1, 307 +/- 29 nM) compared to vehicle-treated controls (Group 4, 152 +/- 6 nM, P < 0.05). Neither calcium antagonist altered ventricular function or [Ca(2+)](i) in sham burns (Groups 5 and 6). In contrast, antagonists given after burn injury reduced cardiomyocyte [Ca(2+)](i) (Group 2, dantrolene-treated burns: 196 +/- 8 nM, and Group 3, diltiazem treated burns: 216 +/- 8 nM) and improved cardiac performance compared to that measured in burns given FR alone. Our data suggest that calcium antagonists given after burn trauma restored intracellular Ca(2+) homeostasis, decreased cardiac cell injury, and improved cardiac contractile function.

Animals↗

PubMed1999

Extended X-ray absorption fine structure studies on periplasmic and intracellular molybdenum-binding proteins.

We have studied the molybdenum K-edge X-ray absorption spectra of Mo bound in the Mo-binding proteins Mop from Haemophilus influenzae, ModG from Azotobacter vinelandii and the Escherichia coli ModE transcriptional regulatory protein, and compared them with the absorption spectra of A. vinelandii ModA and monomeric molybdate. Pre-edge and extended fine structure data indicate that the Mo-binding proteins with molbindin-like domains bind tetrahedral molybdate with a Mo-O distance of 1.76 A. The molbindin subunits or sub-domains represent a novel protein fold that is used by proteins with distinct functions to bind molybdate in the cytoplasm. The high specificity of the proteins for molybdenum does not depend on a change of coordination number or geometry.

Azotobacter vinelandii↗