Detection of Trypanosoma cruzi infection by PCR in Canis lupus familiaris and their ectoparasites in Chile.

Chronic Chagas disease affects humans and animals, involving rural and urban inhabitants. Dogs participate in the maintenance and transmission of Trypanosoma cruzi. The objective of this study was to evaluate the presence of T. cruzi in dogs and their ticks and fleas, in a rural area of Central Chile. Trypanosoma cruzi was detected by PCR both in dogs and ectoparasites. From the blood samples obtained, 57% were infected by T. cruzi, 5.4% of the ticks detected were positive, and all fleas were negative. Additionally, we performed electrocardiograms and found supraventricular arrhythmia in 44% of T. cruzi-positive dogs. Nevertheless, their risk for supraventricular arrhythmias was not higher in infected versus noninfected dogs. Considering the detected infection levels, dogs act as T. cruzi hosts in Central Chile, and ticks could be used as an indicator of infection when blood samples are not available. However, at this point, there is no indication that these ticks could pass on the parasite to another host. Periodic ectoparasitic treatment of pets should reduce the chance of vectorial transmission of T. cruzi and improve canine health; however, this is an uncommon practice among rural communities, so governmental programs are encouraged to tackle this problem.

The 2020 International Alliance for the Control of Scabies Consensus Criteria for the Diagnosis of Scabies.

BACKGROUND: Scabies is a common parasitic skin condition that causes considerable morbidity globally. Clinical and epidemiological research for scabies has been limited by a lack of standardization of diagnostic methods. OBJECTIVES: To develop consensus criteria for the diagnosis of common scabies that could be implemented in a variety of settings. METHODS: Consensus diagnostic criteria were developed through a Delphi study with international experts. Detailed recommendations were collected from the expert panel to define the criteria features and guide their implementation. These comments were then combined with a comprehensive review of the available literature and the opinion of an expanded group of international experts to develop detailed, evidence-based definitions and diagnostic methods. RESULTS: The 2020 International Alliance for the Control of Scabies (IACS) Consensus Criteria for the Diagnosis of Scabies include three levels of diagnostic certainty and eight subcategories. Confirmed scabies (level A) requires direct visualization of the mite or its products. Clinical scabies (level B) and suspected scabies (level C) rely on clinical assessment of signs and symptoms. Evidence-based, consensus methods for microscopy, visualization and clinical symptoms and signs were developed, along with a media library. CONCLUSIONS: The 2020 IACS Criteria represent a pragmatic yet robust set of diagnostic features and methods. The criteria may be implemented in a range of research, public health and clinical settings by selecting the appropriate diagnostic levels and subcategories. These criteria may provide greater consistency and standardization for scabies diagnosis. Validation studies, development of training materials and development of survey methods are now required. What is already known about this topic? The diagnosis of scabies is limited by the lack of accurate, objective tests. Microscopy of skin scrapings can confirm the diagnosis, but it is insensitive, invasive and often impractical. Diagnosis usually relies on clinical assessment, although visualization using dermoscopy is becoming increasingly common. These diagnostic methods have not been standardized, hampering the interpretation of findings from clinical research and epidemiological surveys, and the development of scabies control strategies. What does this study add? International consensus diagnostic criteria for common scabies were developed through a Delphi study with global experts. The 2020 International Alliance for the Control of Scabies (IACS) Criteria categorize diagnosis at three levels of diagnostic certainty (confirmed, clinical and suspected scabies) and eight subcategories, and can be adapted to a range of research and public health settings. Detailed definitions and figures are included to aid training and implementation. The 2020 IACS Criteria may facilitate the standardization of scabies diagnosis.

Determination of the welfare status of freeroaming dogs in two urban centres in Chile. / Determinación del estado de bienestar en perros callejeros de dos centros urbanos de Chile.

Free-roaming dogs are not only a public health and ethical problem, they are also an environmental and economic one. Although the general belief is that freeroaming dogs are not in good condition, there have been insufficient studies in Chile to address and analyse the issue. The objective of this research was to assess the welfare of free-roaming dogs in the centre of the cities of Santiago and Valparaíso. The evaluation was carried out using an observational method and assessed a total of 554 dogs. The following variables were analysed: body condition, motor impairment, skin condition, respiratory disease, reaction to humans and other variables. In addition, spatial distribution was considered, and age, sex and social behaviour were estimated. When the results were analysed in terms of frequency, it was found that, in Valparaíso (n = 204), 37% of the dogs assessed had compromised welfare (poor or fair), while 63% had positive welfare (good or optimal). In contrast, in Santiago (n = 350), only 21.7% had compromised welfare, while 78.3% had positive welfare. With respect to social behaviour, 55% of the dogs assessed in Valparaíso and 68% of those assessed in Santiago led a solitary lifestyle. Although most of the individuals were in good condition, a high percentage were unable to meet the requirements for them to live in harmony with their environment.

Les chiens de rue ou errants posent des problèmes non seulement de santé publique et d'éthique, mais aussi environnementaux et économiques. La condition générale de ces chiens est intuitivement perçue comme n'étant pas bonne mais au Chili le sujet n'a guère fait l'objet d'analyses approfondies. Les auteurs présentent les résultats d'une étude visant à évaluer le bien-être animal des chiens errants du centre-ville de Santiago et de Valparaíso. Cette évaluation a reposé sur l'observation de 554 chiens au total et sur l'analyse d'un certain nombre de variables dont la condition physique, les difficultés motrices, l'état de la peau et du pelage, la présence de maladies respiratoires et les réactions face à l'être humain. La distribution spatiale des chiens examinés a été consignée, ainsi que l'âge estimé, le sexe et le comportement social de chaque animal. Les résultats ont été analysés en termes de fréquence. À Valparaíso, les observations ont fait état d'un niveau de bien-être compromis (allant de mauvais à médiocre) chez 37 % des chiens (n = 204) et d'un pourcentage de 63 % de chiens présentant un niveau de bien-être satisfaisant (bon à optimal). En revanche, à Santiago (n = 350), 21 % seulement des chiens observés présentaient un état de bien-être compromis tandis que 78,3 % présentaient un état de bien-être satisfaisant. En ce qui concerne le comportement social, 55 % des chiens observés à Valparaíso et 68 % de ceux observés à Santiago avaient un mode de vie solitaire. Si la majorité des chiens se trouvaient en bon état physique, un pourcentage élevé d'entre eux ne parvenaient pas à satisfaire les besoins leur permettant de vivre en équilibre avec leur environnement.

Los perros callejeros constituyen no solo un problema de salud pública y ético, sino también, medioambiental y económico. Si bien se cree que estos perros no se encuentran en buenas condiciones, en Chile los estudios que abordan y analizan este tema son insuficientes. El objetivo de esta investigación fue evaluar el bienestar en perros callejeros en el centro de las ciudades de Santiago y Valparaíso. La evaluación se realizó a través de un método observacional en un total de 554 perros, y se analizaron las siguientes variables: condición corporal, dificultad motora, estado de la piel, enfermedad respiratoria, reacción hacia el humano y otras variables. Además, se consideró la distribución espacial y se estimó la edad, el sexo y la conducta social. Los resultados fueron analizados en términos de frecuencia, y se observó que en Valparaíso (n = 204), el 37% de los perros presentaba un bienestar comprometido (malo - escaso) mientras que el 63% presentaba un bienestar favorable (bueno u óptimo). En cambio, en Santiago (n = 350), sólo un 21,7% presentaba un estado de bienestar comprometido mientras que el 78,3% presentaba un estado de bienestar favorable. Asimismo, respecto a la conducta social, un 55% de los perros evaluados en Valparaíso y un 68% de los evaluados en Santiago mostraron un estilo de vida solitario. Si bien los individuos en su mayoría se encontraban en buenas condiciones, un alto porcentaje no lograba satisfacer las necesidades para estar en equilibrio con su entorno.

Clast shape-fabric analysis: A comprehensive and efficient methodology to measure particle-orientation data in solid and loose volcaniclastic deposits.

Fabric analysis is essential for understanding the evolution of volcaniclastic deposits. Here we present a comprehensive and efficient methodology, called "Clast shape-fabric analysis," which is part of the Quantitative Textural Analysis (QTA). This methodology combines high-resolution image analysis techniques with geospatial data processing tools. The fabric of a deposit refers to the three-dimensional orientation of the particles with respect to space, where the degree of iso-orientation of the major axes of the particles is taken into account. The process begins with the collection of oriented samples in the field. Then, in the laboratory, the samples are processed to obtain high-resolution images. The final stage involves the analysis of these images using the FabricS program, which combines image processing techniques and circular statistics. An application of the method was made at the Joya Honda Maar in Mexico, where shape-fabric analysis was used to identify the emission centers of pyroclastic materials. In summary, the "Clast shape-fabric analysis" is a reliable, low-cost and high-potential methodology that can be applied in several geoscientific disciplines and other areas of scientific research.•New Methodology for shape-fabric analysis is presented.•The methodology involves field work, laboratory work and image analysis.•Identification of particle orientations in volcaniclastic deposits.

Biological and genetic aspects of crosses between phylogenetically close species of Mexican triatomines (Hemiptera: Reduviidae).

Reproductive isolation between Triatoma mexicana (Herrich-Scaeffer) and the six species of the Phyllosoma complex, belonging to the genus Meccus, was examined by analyzing the fertility of experimental hybrids. The percentage of couples with offspring was highest in the set of crosses between Meccus phyllosomus (Burmeister) and T. mexicana, and lowest in those between Meccus pallidipennis (Stål) and Meccus bassolsae (Alejandre-Aguilar, Nogueda-Torres, Cortés-Jiménez, Jurberg, Galvão, and Carcavallo) with respect to T. mexicana. Crosses between Meccus mazzottii (Usinger), Meccus picturatus (Usinger), and Meccus longipennis (Usinger) and T. mexicana had scarce offspring, whereas offspring from crosses between M. phyllosomus and T. mexicana were abundant. However, all F1 nymphs died in different instars before reaching sexual maturity. The biological evidence in this study suggests that different degrees of reproductive isolation exist between T mexicana and the different species of Meccus. Our data also support the current status of T. mexicana as a separate species.

Wastewater treatment using a novel bioreactor with submerged packing bed of polyethylene tape.

The performance of a novel aerobic bioreactor with a specially designed submerged packing bed of high specific surface area density, made of polyethylene tape, was studied for the treatment of domestic wastewater. The reactor has a volume of 0.71 m(3) and the specific area of the packing bed was 1,098 m(2)/m(3). The operation was performed with and without effluent recycling, applying different organic loads in the range of 4.0-17.6 g COD m(-2) d(-1). No back-washings were carried out. Overall BOD(5) removals of 90-95% were obtained with organic loads of 4.0-17.6 g COD m(-2) d(-1) and HRT of 0.2-1.1 h. Overall TN removal of 69-72% was obtained at loads of 0.8-4.6 g TN m(-2) d(-1) when effluent recycling was used. The reactor allowed obtaining high quality water for urban reuse and demonstrated an effective process performance and resistance to load variations. The developed biofilm was completely penetrated by the organic matter, ammonia and oxygen, providing high removal rates. Large biomass quantities, up to 13 g dry VS/m(2), were reached in the reactor and the determined sludge yield coefficient was relatively low, of 0.25 g VSS/g COD. These results allow obtaining compact treatment systems with low sludge production and make the technology a suitable option for small wastewater treatment plants.

Treatment of cane sugar mill wastewater in an upflow anaerobic sludge bed reactor.

The performance of a mesophilic UASB reactor was studied for the treatment of sugar cane mill wastewater previously pre-treated for solid separation. The experimental work was carried out in a reactor with 80 L total volume. Four organic loads were applied and the process performance was evaluated during two months for each experimental stage. Removal efficiencies higher than 90% were obtained with organic loads up to 16 kg COD m(-3) d(-1). Stable process performance and high biogas production were obtained. The COD removal rate increased substantially with the load increase to 24 kg COD m(-3) d(-1). However, the obtained removal was of only 78-82%, which can be attributed to the accumulation of volatile organic acids. The kinetic coefficients were obtained using first order model for the substrate removal rate and Monod's equation for bacteria specific growth rate. The UASB reactor is a good option for the biological treatment of pre-treated sugar cane mill wastewaters. The discharge requirements for COD concentration can be accomplished if the reactor is operated at a low organic load of 4 kg COD m(-3) d(-1). At higher loads, an additional biological treatment stage is needed.

The brain-derived neurotrophic factor VAL68MET polymorphism modulates how developmental ethanol exposure impacts the hippocampus.

Prenatal exposure to alcohol causes a wide range of deficits known as fetal alcohol spectrum disorders (FASDs). Many factors determine vulnerability to developmental alcohol exposure including timing and pattern of exposure, nutrition and genetics. Here, we characterized how a prevalent single nucleotide polymorphism in the human brain-derived neurotrophic factor (BDNF) gene (val66met) modulates FASDs severity. This polymorphism disrupts BDNF's intracellular trafficking and activity-dependent secretion, and has been linked to increased incidence of neuropsychiatric disorders such as depression and anxiety. We hypothesized that developmental ethanol (EtOH) exposure more severely affects mice carrying this polymorphism. We used transgenic mice homozygous for either valine (BDNFval/val ) or methionine (BDNFmet/met ) in residue 68, equivalent to residue 66 in humans. To model EtOH exposure during the second and third trimesters of human pregnancy, we exposed mice to EtOH in vapor chambers during gestational days 12 to 19 and postnatal days 2 to 9. We found that EtOH exposure reduces cell layer volume in the dentate gyrus and the CA1 hippocampal regions of BDNFmet/met but not BDNFval/val mice during the juvenile period (postnatal day 15). During adulthood, EtOH exposure reduced anxiety-like behavior and disrupted trace fear conditioning in BDNFmet/met mice, with most effects observed in males. EtOH exposure reduced adult neurogenesis only in the ventral hippocampus of BDNFval/val male mice. These studies show that the BDNF val66met polymorphism modulates, in a complex manner, the effects of developmental EtOH exposure, and identify a novel genetic risk factor that may regulate FASDs severity in humans.

Cytotoxicity and biological capacity of sulfur-free lignins obtained in novel biorefining process.

Lignin is one of the most promising and versatile products obtained in biorefineries due to its diverse therapeutic properties such as antimicrobial, antioxidant and anti-inflammatory activity. However, these properties depend on the source of lignin and the way it was isolated from the biomass. In this study, four different lignins are compared (extracted with Aquasolv (ASL1, ASL2), Organosolv (OSL) and Alkali (ALK) processes) for their cellular antioxidant capacity, anti-diabetic activity, free radical scavenging and cytotoxicity. Alkali and Organosolv lignins showed the highest antioxidant capacity 1159.815 µmol TE g-1 and 1463.415 µmol TE g-1, respectively, in agreement with their highest amount of free -OH groups. Additionally, OSL showed the highest inhibition in the antidiabetic assay followed by ASL1 with values for α-amylase of 3.6 mg/ml and 4.3 mg/ml respectively, and α-glucosidase 1.6 mg/ml and 2.5 mg/ml correspondingly. Nevertheless, cell-based assays revealed that ASL has the lowest cytotoxic effect in Caco-2 cells and, thus, is 10 times less cytotoxic than Alkali and OSL. This work suggests the applicability of ASL for high value applications such as cosmetics or pharmaceuticals. INDUSTRIAL RELEVANCE: Industrially, Liquid Hot Water (LHW) and Organosolv processes may appear as promising biorefining technologies in the following years. Lignin produced is free of sulfur, can be labelled as clean and environmentally-friendly and in this study, was proven that LHW lignin is non cytotoxic. The findings in this paper showed that different sources of lignin can be used in product formulation for life science purposes, thus opening a broad spectrum of possibilities for lignin valorisation in biorefineries.

Comparison of bioreactors with different kinds of submerged packed beds for domestic wastewater treatment.

The performance of aerobic submerged packed bed reactors was studied for the treatment of domestic wastewater using different kinds of packing materials with high specific areas (760-1,200 m(2)/m(3)). The tested materials were ceramic spheres, crushed tezontle, grains of high density polyethylene (HDPE), of low density polyethylene (LDPE) and of polypropylene (PP), cubes of polyurethane (PU) and polyethylene tape (SESSIL). The bioreactors were operated in continuous regime, applying organic loads in the range of 0.8-6.0 g COD.m(-2).d(-1). The obtained specific COD removal rates were very similar in all the reactors when they were operated at organic loads up to 2.0 g COD.m(-2).d(-1), after which differences in effectiveness appeared and the best results were determined in the reactors with SESSIL, LDPE and PU. Very low TSS, O&G and turbidity were obtained in all the effluents. The NH(3)-N and TN removals were dependent on the dissolved oxygen (DO) concentration and the removals at DO of 5 mg/l were 84-99% and 61-74% respectively. The best removals were determined in the reactors with PU, SESSIL and LDPE. The reactor with tezontle had also a good performance when operated with loads up to 1.0 g TN.m(-2).d(-1). The best phosphate removals (38-49%) were obtained in the reactors with PU, tezontle, ceramic sheres and SESSIL.

Long-term Reductions in the Population of GABAergic Interneurons in the Mouse Hippocampus following Developmental Ethanol Exposure.

Developmental exposure to ethanol leads to a constellation of cognitive and behavioral abnormalities known as Fetal Alcohol Spectrum Disorders (FASDs). Many cell types throughout the central nervous system are negatively impacted by gestational alcohol exposure, including inhibitory, GABAergic interneurons. Little evidence exists, however, describing the long-term impact of fetal alcohol exposure on survival of interneurons within the hippocampal formation, which is critical for learning and memory processes that are impaired in individuals with FASDs. Mice expressing Venus yellow fluorescent protein in inhibitory interneurons were exposed to vaporized ethanol during the third trimester equivalent of human gestation (postnatal days 2-9), and the long-term effects on interneuron numbers were measured using unbiased stereology at P90. In adulthood, interneuron populations were reduced in every hippocampal region examined. Moreover, we found that a single exposure to ethanol at P7 caused robust activation of apoptotic neurodegeneration of interneurons in the hilus, granule cell layer, CA1 and CA3 regions of the hippocampus. These studies demonstrate that developmental ethanol exposure has a long-term impact on hippocampal interneuron survivability, and may provide a mechanism partially explaining deficits in hippocampal function and hippocampus-dependent behaviors in those afflicted with FASDs.

The prothrombotic state associated with obesity-induced hypertension is reduced by cocoa and its main flavanols.

BACKGROUND: Little is known about the effects of cocoa and its main flavanols on the prothrombotic state associated with the development of hypertension in diet-induced obesity models. PURPOSE: To evaluate the effects of cocoa powder, cocoa extract and their main flavanols on plasma biomarkers related to impaired coagulation and fibrinolysis and its association with hypertension and obesity-related metabolic disorders in rats fed a hypercaloric diet. METHODS: Male Wistar rats were randomly assigned to 7 treatment groups (n = 7): normal diet (ND); hypercaloric diet control group (HCD); HCD + cocoa powder (CO); HCD + cocoa extract (CO-EX); HCD + (-)-epicatechin (EPI); HCD + (+)-catechin (CAT); and HCD + procyanidin B2 (PB2). Blood pressure was measured using the tail-cuff method (week 7). At the end of the experimental period (week 8), rats were sacrificed and blood samples were collected immediately for coagulation and biochemical analyses. RESULTS: Oral administration of CO, CO-EX and their main flavanols significantly decreased plasma biomarkers related to impaired coagulation and fibrinolysis (vWF, FVIII, fibrinogen and PAI-1) in rats fed a hypercaloric diet. These effects were associated with decreased systolic and diastolic blood pressure, aortic oxidative stress (MDA levels) and improvement of dyslipidemia, insulin resistance and circulating markers of inflammation (TNF-α, IL-6 and CRP) compared to the HCD group. CONCLUSION: Our results showed that cocoa and its main flavanols may improve endothelial dysfunction and exert their antihypertensive effects by decreasing the prothrombotic state in rats fed a hypercaloric diet. Moreover, improvement of obesity-related metabolic disorders may also contribute to their BP-lowering effect.

Transmission Capacity of Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae) by Three Subspecies of Meccus phyllosomus (Heteroptera: Reduviidae) and Their Hybrids.

Three behaviors of epidemiological importance: the time lapse for the onset of feeding, actual feeding, and defecation time for Meccus phyllosomus pallidipennis (Stål), Meccus phyllosomus longipennis (Usinger), Meccus phyllosomus picturatus (Usinger), and their laboratory hybrids were evaluated in this study. The mean time lapse for the beginning of feeding was between 0.5 and 8.3 min considering all instars in each cohort, with highly significant differences only among fifth-instar nymphs, females, and males of M. p. pallidipennis and M. p. longipennis relative to the hybrid cohorts. Four hybrid (LoPa [M. p. longipennis and M. p. pallidipennis] and LoPi [M. p. longipennis and M. p. picturatus] and their reciprocal experimental crosses) cohorts had similar mean feeding times to one of the parental subspecies, but longer than the other one. The remaining hybrid cohort (PaPi [M. p. pallidipennis and M. p. picturatus]) had longer feeding times than both of its parental subspecies. The specimens of the LoPa and LoPi hybrid cohorts defecated faster than the respective instars of the three parental cohorts. With exception of first- and fifth-instar nymphs, PaPi cohorts defecated faster than the remaining seven cohorts. More than 60% of defecation events occurred during feeding in the six hybrid cohorts. Our results indicate that hybrid cohorts have more potential to acquire infection and transmit Trypanosoma cruzi Chagas than their parental cohorts.

Calcium uptake, release and ryanodine binding in melanosomes from retinal pigment epithelium.

High levels of calcium have been reported in pigmented tissues of the vertebrate eye, such as retinal pigment epithelium (RPE). Melanin granules also have high calcium concentrations, suggesting that melanin granules may be a calcium reservoir. Here we characterized the uptake and release of calcium in a pure melanosomal fraction obtained from frog RPE. Melanosomes take up 45Ca by a saturable system with an apparent KM of 0.5 mM. About 40% of 45Ca accumulation was insensitive to low temperature. 45Ca uptake was not affected by verapamil, nifedipine, dantrolene, vanadate, thapsigargin or cyclopiazonic acid, but it was reduced by 50% by ruthenium red, and increased by the ionophore A23187 and nigericin. Release of 45Ca-loaded was stimulated by caffeine and inositol 1,4,5 trisphosphate (IP3). Caffeine stimulated release of calcium was blocked by either ryanodine or ruthenium red, but calcium released by IP3 was not affected by heparin. No binding of 3H-IP3 was observed. The 3H-ryanodine binding sites exhibited a KB of 1.3 nM and a Bmax of 12.1 fmol/mg protein. Thus, our results suggest that melanosomes may function as intracellular organelles that regulate calcium concentration in RPE.

Evaluation of the role of recA protein in plant virulence with recA mutants of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris NRRL B1459 recA mutants were isolated by recombination with an interrupted Rhizobium etli recA gene and selection of double recombinants. The mutants were impaired in homologous genetic recombination and in DNA repair as judged by their sensitivity to methyl-methane-sulfonate and to UV irradiation; these defects are complemented in trans by the R. etli recA gene. Virulence of X. campestris pv. campestris NRRL B1459 to cabbage is considerably diminished by the recA mutation. The recA mutation is not correlated with the frequency of occurrence of a genetic rearrangement that affects chemotaxis, plant virulence, and xanthan gum production.

The Azotobacter vinelandii alg8 and alg44 genes are essential for alginate synthesis and can be transcribed from an algD-independent promoter.

A 2.8-kb DNA region, located immediately downstream of algD, contains the A. vinelandii alg8 and alg44 genes, whose sequences are highly homologous to those of the corresponding Pseudomonas aeruginosa genes. These genes occur on a transcript that does not include algD, and are transcribed from a promoter different from that transcribing algD; this is the fourth promoter described within the alginate biosynthetic gene cluster. alg8 and alg44 mutants were constructed and shown to be completely impaired in alginate production. Alg8 shares 28.20% identity and 38.09% similarity to Azorhizobium caulinodans NodC, a glycosyl transferase catalyzing the formation of beta-1,4 linkages. A topological model is predicted, which supports the idea of Alg8 being the polymerase responsible for alginate synthesis.

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria.

Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.

Life-supporting pig-to-primate renal xenotransplantation using genetically modified donors.

BACKGROUND: In order to circumvent the complement-mediated hyperacute rejection of discordant xenografts, a colony of pigs transgenic for the human regulator of complement activity, human decay-accelerating factor (hDAF), has been produced. METHODS: Seven kidneys from hDAF transgenic pigs and six kidneys from nontransgenic control pigs were transplanted into cynomolgus monkeys; both native kidneys were removed during the same operation. The recipient animals were immunosuppressed with cyclosporine, steroids, and cyclophosphamide. RESULTS: In the transgenic group, the median survival time was 13 days (range, 6-35 days); the median survival time in the control group was 6.5 days (range, 0.3-30 days). There were no cases of hyperacute rejection in the transgenic group, and the two longest-surviving kidneys in this group showed no evidence of rejection on histological examination. In contrast, all control kidneys underwent antibody-mediated rejection, one demonstrating hyperacute rejection and the others acute vascular rejection. CONCLUSION: This study demonstrates that (i) a kidney from an hDAF transgenic pig can support the life of a primate for up to 35 days (and also shows the basic physiological compatibility between the pig and nonhuman primate); (ii) nontransgenic kidneys are not routinely hyperacutely rejected; and (iii) the presence of hDAF on the kidney confers some protection against acute vascular rejection. Improved immunosuppression and immunological monitoring may enable extended survival.

Long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts.

BACKGROUND: Recently, there has been a resumed interest in clinical xenotransplantation using pig organs. However, no data are available yet regarding the capacity of porcine organs to sustain the life of a primate beyond the first month. We have attempted to obtain long-term survival of nonhuman primates using human decay-accelerating factor (hDAF) transgenic pig organs and an immunosuppressive strategy particularly aimed at neutralizing the humoral component of the immune response. METHODS: hDAF transgenic or control kidneys were transplanted into 14 bilaterally nephrectomized cynomolgus monkeys (Macaca fascicularis) that underwent splenectomy and were immunosuppressed with cyclosporine A, cyclophosphamide, and steroids. All animals also received recombinant erythropoietin. Postoperatively, the primates were monitored daily. Laboratory evaluations included serum biochemistry, hematology, and measurements of hemolytic antipig antibodies. To assess the role of splenectomy in the control of humoral response, historical data were also used from a group of monkeys (n=7) that received the same immunosuppressive regimen and an hDAF transgenic porcine kidney but did not have splenectomy or receive recombinant erythropoietin. RESULTS: This immunosuppressive approach obtained the longest survival time (78 days) described to date of a primate receiving a life-supporting porcine renal xenograft. Furthermore, four of nine animals in this series survived for 50 days or more. Most biochemical measurements in this study (including plasma urea, creatinine, sodium, and potassium concentrations) remained within normal ranges for several weeks in all of the longest-surviving animals. CONCLUSIONS: Normalization of renal function (urea and creatinine) in primate recipients of porcine renal xenografts suggests that pig kidneys may be suitable for future clinical xenotransplantation. Additional immunosuppressive approaches, specifically designed to prevent humorally mediated immunological damage, should be explored to further prolong survival of primates that have received porcine xenografts.