Deep cutaneous mycoses in kidney transplant recipients: Diagnostic and therapeutic challenges.

Deep cutaneous mycoses (DCMs) are rare infections that extend throughout the dermis and subcutis, often occurring after inoculation with pathogenic fungi. Trends toward a growing incidence have been observed that may be partially related to an increasing population of solid organ transplant patients. The aim of this study is to describe the diagnostics and the outcomes of DCM among kidney transplant recipients so as to optimize their management. We performed a retrospective review of cases of DCM occurring among kidney transplant recipients in our institution over 12 years. Twenty cases were included. Lesions were only located on the limbs and presented mainly as single (10/20, 50%) nodular lesions (15/20, 75%), with a mean size of 3 cm. Direct mycological examination was positive for 17 patients (17/20, 85%) and the cultures were consistently positive. Thirteen different fungal species were observed, including phaehyphomycetes (n = 8), hyalohyphomycetes (n = 3), dermatophytes (n = 1), and mucorale (n = 1). The (1-3) beta-D-glucan antigen (BDG) was also consistently detected in the serum (20/20, 100%). Systematic imaging did not reveal any distant infectious lesions, but locoregional extension was present in 11 patients (11/14, 79%). Nineteen patients received antifungal treatment (19/20, 95%) for a median duration of 3 months, with surgery for 10 (10/20, 50%). There is a great diversity of fungal species responsible for DCMs in kidney transplant recipients. The mycological documentation is necessary to adapt the antifungal treatment according to the sensitivity of the species. Serum BDG positivity is a potentially reliable and useful tool for diagnosis and follow-up.

Advances in Organ and Tissue Xenotransplantation.

End-stage organ failure can result from various preexisting conditions and occurs in patients of all ages, and organ transplantation remains its only treatment. In recent years, extensive research has been done to explore the possibility of transplanting animal organs into humans, a process referred to as xenotransplantation. Due to their matching organ sizes and other anatomical and physiological similarities with humans, pigs are the preferred organ donor species. Organ rejection due to host immune response and possible interspecies infectious pathogen transmission have been the biggest hurdles to xenotransplantation's success. Use of genetically engineered pigs as tissue and organ donors for xenotransplantation has helped to address these hurdles. Although several preclinical trials have been conducted in nonhuman primates, some barriers still exist and demand further efforts. This review focuses on the recent advances and remaining challenges in organ and tissue xenotransplantation.

Characteristics, mortality, associated variables with death, and therapeutic response among HIV-positive, solid organ transplant (SOT), and non-HIV-positive/non-transplant (NHNT) patients with cryptococcosis: First multicenter cohort study in Brazil.

Cryptococcosis is traditionally associated with immunocompromised patients but is increasingly being identified in those without the human immunodeficiency virus (HIV) or other immunocompetent individuals. We aim to describe the characteristics, mortality, and associated variables with death among hospitalized patients with cryptococcosis in Brazil. This is the first multicenter retrospective cohort study conducted in seven public tertiary Brazilian hospitals. A total of 384 patients were included; the median age was 39 years and 283 (73.7%) were men. In all, 304 HIV-positive were hosts (79.2%), 16 (4.2%) solid organ transplant (SOT), and 64 (16.7%) non-HIV-positive/non-transplant (NHNT). Central nervous system (CNS) cryptococcosis had a significantly higher number across disease categories, with 313 cases (81.5%). A total of 271 (70.6%) patients were discharged and 113 (29.4%) died during hospitalization. In-hospital mortality among HIV-positive, SOT, and NHNT was 30.3% (92/304), 12.5% (2/16), and 29.7% (19/64), respectively. Induction therapy with conventional amphotericin B (AMB) mainly in combination with fluconazole (234; 84.2%) was the most used. Only 80 (22.3%) patients received an AMB lipid formulation: liposomal (n = 35) and lipid complex (n = 45). Most patients who died belong to the CNS cryptococcosis category (83/113; 73.4%) when compared with the others (P = .017). Multivariate analysis showed that age and disseminated cryptococcosis had a higher risk of death (odds ratio [OR], 1.03; 95% confidence interval [CI], 1.01-1.05; P = .008 and OR, 1.84; 95% CI, 1.01-3.53; P = .048, respectively). Understanding the epidemiology of cryptococcosis in our settings will help to recognize the burden and causes of mortality and identify strategies to improve this scenario.

This multicenter cohort study included 384 hospitalized individuals with cryptococcosis in Brazil. Most individuals were men (74%), HIV-positive (79%), had central nervous system involvement (82%), and received conventional amphotericin plus fluconazole (84%). In-hospital mortality was high (29%).

Evolution of haematopoietic cell transplantation for canine blood disorders and a platform for solid organ transplantation.

Pre-clinical haematopoietic cell transplantation (HCT) studies in canines have proven to be invaluable for establishing HCT as a highly successful clinical option for the treatment of malignant and non-malignant haematological diseases in humans. Additionally, studies in canines have shown that immune tolerance, established following HCT, enabled transplantation of solid organs without the need of lifelong immunosuppression. This progress has been possible due to multiple biological similarities between dog and mankind. In this review, the hurdles that were overcome and the methods that were developed in the dog HCT model which made HCT clinically possible are examined. The results of these studies justify the question whether HCT can be used in the veterinary clinical practice for more wide-spread successful treatment of canine haematologic and non-haematologic disorders and whether it is prudent to do so.

Transplantation Into the Mouse Ovarian Fat Pad.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

A model for cryobanking female germplasm in Japanese quail (Coturnix japonica).

Cryopreservation and transplantation of ovarian tissue can be used for cryobanking female germplasm of avian species. Here we report a model of cryobanking ovarian tissue of Japanese quail. Ovaries of 1-wk-old quail chicks from the recessive white-breasted (WB) line were cryopreserved using a vitrification method. The cryopreserved tissue was warmed and transplanted orthotopically into ovariectomized, 1-wk-old female quail chicks from the homozygous wild-type QO line. At sexual maturation, recipients were mated with WB males and the plumage coloration was used as a marker to determine the origin of their ovaries. Seven of the 15 recipients laid eggs and produced donor-derived offspring, and 5 produced 100% donor-derived offspring. No difference was seen in egg production between recipients and untreated birds. The high efficiency of the vitrification and transplantation procedures in preserving and recovering quail ovarian tissue shows that this model can be used for maintaining commercial and experimental quail strains and may benefit other poultry species and endangered avian species.

Multiple ovarian transplants to rescue a transgenic line of mice.

Transgenic mice are useful tools for studying gene function and regulation but can be difficult to successfully breed. To 'rescue' transgenic lines that are difficult to propagate, researchers use a variety of techniques. One method is ovarian transplant, in which researchers remove ovaries from a donor transgenic mouse, cryopreserve the ovarian tissue, transplant this tissue into histocompatible female mice and breed these recipient females. Though it is a useful technique, cryopreservation can potentially damage ovarian tissue, which could reduce fertility. In this article, the authors describe how they carried out ovarian transplants without cryopreservation to rescue a line of transgenic C57BL/6 mice. Other researchers who have experience with mouse reproductive surgery should be able to use this technique to rescue infertile transgenic lines of mice.

Development of perioperative care for pigs undergoing laryngeal transplantation: a case series.

Pigs are ideal animal models for airway surgical research, facilitating the successful translation of science into clinical practice. Despite their ubiquitous use, there is a paucity of information on the perioperative care of pigs, especially for major procedures. In a series of experiments to investigate laryngeal transplantation, we combined veterinary and medical experience to develop protocols for perioperative management of pigs, including high dependency care. Novel airway management methods were developed. A pain scoring system was used to direct analgesia use. Fluid balance and electrolytes were monitored closely. Recent animals received a central venous line via the femoral vein two days prior to transplantation to facilitate blood sampling and drug delivery. Intensive monitoring and airway management were required to ensure a successful outcome. Methods for optimal perioperative care are proposed. These results will help future groups wishing to use pigs in airway research, will reduce numbers of animals used and improve animal welfare.

Long-term production of donor-derived offspring from chicken ovarian transplants.

A surgical technique for orthotopic transplantation of ovarian tissue was recently developed for chickens, and short-term production of donor-derived offspring from the transplants was demonstrated. The present study evaluated the long-term reproductive function of the transplanted ovaries. In addition, ovarian tissue was transplanted into chicks from eggs that had been treated with busulfan (BU) after 24 h of incubation to reduce the growth of host germ cells. The success rate of ovarian transplantation was not different between BU- and non-BU-treated birds, but the ratio of donor-derived to host-derived offspring was significantly higher in the BU-treated birds. Over the 18-mo period each successfully transplanted hen produced up to 396 eggs with between 50 to 98% of donor-derived progeny. These results demonstrate that when transplanted ovaries attach and develop they can maintain their reproductive function for an extended period of time. These transplantation techniques provide a unique opportunity for development of a practical, efficient protocol for the cryopreservation of germplasm of chickens and may provide an opportunity for regenerating populations of wild birds.

Offspring produced from orthotopic transplantation of chicken ovaries.

The loss of avian genetic variation and the threat of disease lend urgency to the cryopreservation of remaining poultry stocks. However, techniques for freezing ova and embryos are not available for birds, and the recovery of genetic material has been a major obstacle to cryopreservation. To overcome this problem, we transplanted chicken ovarian tissue just after hatch with or without subsequent treatment of the recipient with an immunosuppressant. Nine of 12 hens in the nonimmunosuppressed group and 6 of 9 birds in the immunosuppressed group produced eggs, whereas 3 hens in each group produced donor-derived offspring. These results suggest that transplantation of ovarian tissue of chickens is possible if performed just after hatch. This finding should allow efficient cryopreservation of female germ cells in chickens with regeneration in live birds. In addition, ovarian transplantation could be useful for studies in genetics or developmental biology or could provide convenient access to the female germline for genetic manipulation.

The technique of orthotopic ovarian transplantation in the chicken.

A surgical technique has been established for orthotopic transplantation of ovarian tissue in newly hatched chickens. In these trials survivability of the chicks after surgical manipulation was 100%. The size and orientation of the ovary at 2 wk of age suggested that the graft was attached and had undergone development. This technique should allow the development of a cryopreservation protocol of chicken ovarian tissue for the conservation of poultry genetic material.

Tolerability of cyclosphosphamide and methotrexate induction immunosuppression in nonhuman primates.

Transplantation in nonhuman primates, in particular using solid organs from porcine donors, requires an efficacious induction immunosuppression. Besides biologicals, the low molecular weight drugs used include cyclophosphamide (CyP) and methotrexate (MTX). As these compounds generally have a narrow therapeutic window, we performed tolerability studies in baboons and cynomolgus monkeys, with/without maintenance immunosuppressants such as cyclosporine A, everolimus, mycophenolate sodium and FTY720. In both species, a four-dose CyP regimen of 40, 20, 30 and 30 mg/kg i.v. on days 1, 2, 4 and 6 is not tolerated, but the regimen is tolerated upon individual adjustment of the third and fourth dose to 18-25 and 8-20mg/kg, respectively, based on white blood cell count. In cynomolgus monkeys, a 5-day course of MTX i.v. at 0.5 mg/(kg d) is well tolerated, but not MTX at 1.0 mg/(kg d); in combination with maintenance immunosuppression, the 0.5mg/(kg d) dose can cause adverse effects. Combinations of CyP and MTX are tolerated using the 5-day course of MTX at 0.25 mg/(kg d) and a four-dose regimen of CyP at 10, 2.5, 7.5 and 7.5 mg/kg. These regimens are tolerated in combination with maintenance immunosuppressants. The data provide base values for investigators using nonhuman primates in experimental studies, particularly in xenotransplantation requiring effective induction immunosuppression that is close to maximum tolerated dose levels.

Advances in biotechnology: new tools in future pig production for agriculture and biomedicine.

Biotechnology in livestock comprises an arsenal of reproductive biotechniques and molecular genetics. While molecular genetics are poorly developed in swine, reproductive techniques are more advanced and applied under field conditions. This review describes three selected examples of our own research to illustrate the implication of biotechnology in future pig reproduction. Sperm sexing technology is now available and can be used to generate piglets of the desired sex by IVF and ICSI. First studies also indicate satisfactory success rates following intrauterine insemination with sexed spermatozoa. Cloning technique and production of transgenic pigs require information about the regulation and time course of gene expression during in vitro production and pre-implantation development. Information on gene expression is scare in porcine embryos. With the exception of transcripts for the oestrogen receptor gene, no mRNA's from the activated porcine genome have been identified. Recent development of cDNA arrays might help to identify a larger amount of genes in single embryos. Remarkable progress has been made in organ transplantation technology. As the demand for human organs is increasing rapidly, the pig might serve as donor of xenotranplants, provided the transmission of zoonoses from the donor animal to the human recipient is prevented, donor organ anatomy and function are compatible, and immunological rejections (HAR, VAR MAC) can be overcome. The most promising strategy is the synthesis of human complement regulatory proteins in the pig. Transgenic pigs have been generated for hDAF or hCD 46 and their hearts have been transplanted into non human primates for up to 90 days. HCD 59 driven by CMV promotor provides significant protection against HAR at least under in vitro and in vivo conditions. Current studies indicate a temporary use in patients within the next 3-5 years. As the microinjection technology to produce transgenic offspring is time consuming and very expensive, nuclear transfer technology provides a possibility for multiplication without going through the germ line with recombination effects. A functional nuclear transfer system will be crucial for xenotransplantation as it is anticipated that the expression of several transgenes will be required.