Author Correction: Consistent success in life-supporting porcine cardiac xenotransplantation.

In this Letter, Mayuko Kurome and Valeri Zakhartchenko have been added to the author list (affiliated with Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany). The author list and 'Author contributions' section have been corrected online; see accompanying Amendment.

Consistent success in life-supporting porcine cardiac xenotransplantation.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1-3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation7.

Uncoupling the widespread occurrence of anti-NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model.

Autoantibodies of the IgG class against N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1-AB) were considered pathognomonic for anti-NMDAR encephalitis. This view has been challenged by the age-dependent seroprevalence (up to >20%) of functional NMDAR1-AB of all immunoglobulin classes found in >5000 individuals, healthy or affected by different diseases. These findings question a merely encephalitogenic role of NMDAR1-AB. Here, we show that NMDAR1-AB belong to the normal autoimmune repertoire of dogs, cats, rats, mice, baboons, and rhesus macaques, and are functional in the NMDAR1 internalization assay based on human IPSC-derived cortical neurons. The age dependence of seroprevalence is lost in nonhuman primates in captivity and in human migrants, raising the intriguing possibility that chronic life stress may be related to NMDAR1-AB formation, predominantly of the IgA class. Active immunization of ApoE-/- and ApoE+/+ mice against four peptides of the extracellular NMDAR1 domain or ovalbumin (control) leads to high circulating levels of specific AB. After 4 weeks, the endogenously formed NMDAR1-AB (IgG) induce psychosis-like symptoms upon MK-801 challenge in ApoE-/- mice, characterized by an open blood-brain barrier, but not in their ApoE+/+ littermates, which are indistinguishable from ovalbumin controls. Importantly, NMDAR1-AB do not induce any sign of inflammation in the brain. Immunohistochemical staining for microglial activation markers and T lymphocytes in the hippocampus yields comparable results in ApoE-/- and ApoE+/+ mice, irrespective of immunization against NMDAR1 or ovalbumin. These data suggest that NMDAR1-AB of the IgG class shape behavioral phenotypes upon access to the brain but do not cause brain inflammation on their own.

Favorable outcome of experimental islet xenotransplantation without immunosuppression in a nonhuman primate model of diabetes.

Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencapsulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression. This strategy should lead to pioneering clinical trials with xenotransplantation for treatment of diabetes and, thereby, represents a previously unidentified approach to efficient cell replacement for a broad spectrum of endocrine disorders and other organ dysfunctions.

CUTANEOUS DEMODICOSIS AND UV-INDUCED SKIN NEOPLASIA IN TWO GOELDI'S MONKEYS (CALLIMICO GOELDII).

Two nonrelated Goeldi's monkeys (Callimico goeldii) from the same enclosure developed multifocal alopecia with hyperkeratotic to ulcerative skin lesions on the lower abdomen and inner thighs. Necropsy samples of the first animal showed hyperplastic dermatitis together with in situ carcinoma and intralesional Demodex organisms. The second monkey developed similar lesions 2.5 yr later. Skin scrapings and biopsies also revealed Demodex mites within hyperplastic dermatitis. Long-term treatment with ivermectin, imidacloprid-moxidectin, and sarolaner resolved the demodicosis but skin lesions progressed to actinic keratosis and carcinoma. Both cutaneous neoplasia and demodicosis are rarely described in New World monkeys and these are the first reported cases in Goeldi's monkeys. Since the animals had access to ultraviolet (UV) light, as recommended for indoor-housed callitrichids, the skin tumors were likely UV-induced and the mites have settled particularly within impaired regions. Thus, apparent demodicosis can indicate cutaneous immunosuppression and might alert caretakers to adjust the UV regime.

TAENIA CRASSICEPS CYSTICERCOSIS IN A NILGIRI LANGUR ( SEMNOPITHECUS JOHNII).

A captive-born adult female Nilgiri langur ( Semnopithecus johnii) developed an edematous swelling of the left thigh and a firm mass around the right ankle joint. The animal also suffered from lethargy and anorexia and was euthanized because of poor general condition. Necropsy revealed that the skeletal muscle of the left thigh had been replaced by a multilocular cystic mass containing numerous sand-grain-sized whitish structures. Small cysts were also present in the lung and the myocardium. The mass of the right ankle joint was histologically consistent with a myxosarcoma. In contrast, the cystic masses from the left thigh, the lung, and the myocardium represented metacestode tissue with evidence of numerous larval cestodes consistent with cysticerci. Cysticerci showed morphological characteristics of Cysticercus longicollis, the larval form of Taenia crassiceps, which was confirmed by genetic analysis. This is the first documented case of a Taenia crassiceps cysticercosis in an Old World monkey species.

Long-term efficient control of SIV infection in macaques is associated with an intact intestinal barrier.

Hallmarks of SIV infection are early depletion of gut CD4 T cells and diminished intestinal integrity. Comprehensive studies on colon biopsies of SIV-infected macaques efficiently controlling infection revealed that in contrast to viremic and failing controllers, elite controllers show preserved CD4 T cells, and low viral load, apoptosis, and inflammation.

Detection of Mycobacterium avium subsp. paratuberculosis in non-human primates.

BACKGROUND: Due to a sporadic occurrence of Mycobacterium avium subsp. paratuberculosis (MAP) in non-human primates (NHP), the susceptibility of different NHP to MAP should be investigated. METHODS: Fecal and tissue samples (ileum, ileocecal lymph node, bone marrow) of 20 animals (seven species) were analyzed by IS900-based PCRs and sequenced. Samples of MAP PCR positive NHP were further cultivated. RESULTS: MAP DNA was detectable in two animals; the ileum of a cottontop tamarin and the bone marrow of a common marmoset. Cultivation of MAP failed. Sequence analysis revealed 100% homology to the MAP-K10 sequence. Pathohistological examinations offered no direct correlation to a MAP infection. CONCLUSIONS: MAP was detected for the first time in a common marmoset. But as both NHP suffered from other diseases, an asymptomatic infection with MAP was assumed. The detection of MAP in the bone marrow might play a role in establishing latent paratuberculosis, as known from tuberculosis.

DETECTION OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN ROCK HYRAXES ( PROCAVIA CAPENSIS) IMPORTED FROM SOUTH AFRICA.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic, progressive, and consecutively fatal enteritis, especially in ruminants. MAP distribution among wildlife is not yet clear. In this study, three wild-born rock hyraxes ( Procavia capensis) had been imported from South Africa to a German zoological garden. During the quarantine period, four young animals were born. The wild-born animals showed symptoms of mild diarrhea shortly after their arrival in the zoological garden, but all routine parasitological and bacteriologic tests performed were negative. Therefore, the animals were additionally tested for MAP infection. MAP DNA was detected by seminested PCR (snPCR) in a pooled fecal sample of the seven animals. Subsequent PCR analysis of the individual feces samples confirmed the excretion of MAP in two rock hyraxes (one wild-born and one born in captivity). Sequence analysis of the corresponding 278-bp amplicons revealed 100% homology to the reference MAP-K10 IS900 sequence. No antibody response against MAP was detected in the individual serum samples. MAP-specific postmortem lesions were not observed by gross pathology and histology, neither after death nor after euthanization of the animals. Nevertheless, MAP was detected by snPCR and culture in the gastrointestinal tract, urogenital tract, cardiovascular system, and/or respiratory system of three other animals of the group (one wild-born and two born in captivity). This study is the first report confirming MAP occurrence in rock hyraxes. Therefore, it is recommended that veterinarians and zoo employees consider rock hyraxes as a possible source of MAP infection for domestic livestock in South Africa and the valuable animal stock of zoological facilities.

Airway hyper-responsiveness in lipopolysaccharide-challenged common marmosets (Callithrix jacchus).

Animal models with a high predictive value for human trials are needed to develop novel human-specific therapeutics for respiratory diseases. The aim of the present study was to examine lung-function parameters in marmoset monkeys (Callithrix jacchus) that can be used to detect pharmacologically or provocation-induced AHR (airway hyper-responsiveness). Therefore a custom-made lung-function device that allows application of defined aerosol doses during measurement was developed. It was hypothesized that LPS (lipopolysaccharide)-challenged marmosets show AHR compared with non-challenged healthy subjects. Invasive plethysmography was performed in 12 anaesthetized orotracheally intubated and spontaneously breathing marmosets. Pulmonary data of R(L) (lung resistance), C(dyn) (dynamic compliance), EF50 (mid-expiratory flow), P(oes) (oesophageal pressure), MV (minute volume), respiratory frequency (f) and V(T) (tidal volume) were collected. Measurements were conducted under baseline conditions and under MCh (methacholine)-induced bronchoconstriction. The measurement was repeated with the same group of animals after induction of an acute lung inflammation by intratracheal application of LPS. PDs (provocative doses) of MCh to achieve a certain increase in RL were significantly lower after LPS administration. AHR was demonstrated in the LPS treated compared with the naïve animals. The recorded lung-function data provide ground for pre-clinical efficacy and safety testing of anti-inflammatory substances in the common marmoset, a new translational NHP (non-human primate) model for LPS-induced lung inflammation.

Distribution of ciliated epithelial cells in the trachea of common marmosets (Callithrix jacchus).

BACKGROUND: The distribution of ciliated cells in the tracheal epithelium of common marmosets was evaluated. METHODS: Light and scanning electron microscopy of tracheal epithelium was performed. RESULTS: Ciliated cells were concentrated in cartilage-free areas and virtually absent in cartilage-supported epithelial regions. CONCLUSIONS: Heterogeneous distribution of ciliated cells in the trachea has to be considered when using animal models for translational respiratory research approaches.

BSE-associated prion-amyloid cardiomyopathy in primates.

Prion amyloidosis occurred in the heart of 1 of 3 macaques intraperitoneally inoculated with bovine spongiform encephalopathy prions. This macaque had a remarkably long duration of disease and signs of cardiac distress. Variant Creutzfeldt-Jakob disease, caused by transmission of bovine spongiform encephalopathy to humans, may manifest with cardiac symptoms from prion-amyloid cardiomyopathy.

Distribution and morphology of Clara cells in common marmosets (Callithrix jacchus).

BACKGROUND: The objective of this investigation was to define the phenotype and spatial distribution of Clara cells within the respiratory tract of common marmosets and to distinguish them from other non-ciliated cells (goblet cells, mixed type secretory cells). METHODS: Non-ciliated cells were identified immunohistochemically using antibodies against Clara cell secretory protein and mucin 5AC. Transmission electron microscopy and scanning electron microscopy were performed to characterize Clara cells ultrastructurally. RESULTS: Clara cells were present throughout the tracheobronchial tree, with lowest numbers in the trachea and highest numbers in bronchioles. Goblet cells and mixed type cells were scarce in the upper conducting airways and virtually absent within bronchioles. Ultrastructurally, Clara cells showed typical apical electron-dense granules and a prominent granular endoplasmatic reticulum. CONCLUSIONS: Clara cells of common marmosets have species-specific morphological characteristics, which suggest grouping the common marmoset phenotypically between primates and rodents.

Atypical cilia in the respiratory tract of common marmosets (Callithrix jacchus) with and without concurrent lung disease.

Ciliated cells of the respiratory epithelium play an essential role in the mechanisms of mucociliary clearance, and ultrastructural alterations of cilia are known to be associated with respiratory disease. The aim of this study was to evaluate the presence of ciliary changes in common marmosets (Callithrix jacchus) of different age groups and to compare healthy animals with animals suffering from subclinical chronic inflammatory pulmonary lesions. Therefore, samples of different sites of the tracheobronchial tree from 24 common marmosets were investigated by transmission electron microscopy. Ciliary alterations were present in all animals and were represented by compound cilia ("bulging/adhesive type"), extramatrix, extratubuli, and disorientation of the microtubular arrangement. Ciliary alterations affected less than 1% of cilia (average 0.06%-0.55%) with no statistically significant differences between age groups, sample localizations, or healthy and sick animals. The study results suggest that ciliary alterations of secondary nature are a common background finding in common marmosets with individual variability in abundance and have to be considered when interpreting ultrastructural data from respiratory studies.

X chromosomal variation is associated with slow progression to AIDS in HIV-1-infected women.

AIDS has changed from a mostly male-specific health problem to one that predominantly affects females. Although sex differences in HIV-1 susceptibility are beyond doubt, the extent to which sex affects the onset and progression of AIDS has remained elusive. Here, we provide evidence for an influence of X chromosomal variation on the course of retroviral infection, both in HIV-1-infected patients and in the rhesus macaque model of AIDS. A two-stage, microsatellite-based GWAS of SIV-infected monkeys revealed MHC class I markers and a hitherto-unknown X chromosomal locus as being associated with a nominal score measuring progression to AIDS (Fisher's exact p < 10(-6)). The X chromosomal association was subsequently confirmed in HIV-1-infected patients with published SNP genotype data. SNP rs5968255, located at human Xq21.1 in a conserved sequence element near the RPS6KA6 and CYLC1 genes, was identified as a significant genetic determinant of disease progression in females (ANOVA p = 8.8 x 10(-5)), but not in males (p = 0.19). Heterozygous female carriers of the C allele showed significantly slower CD4 cell decline and a lower viral load at set point than TT homozygous females and than males. Inspection of HapMap revealed that the CT genotype is significantly more frequent among Asians than among Europeans or Africans. Our results suggest that, in addition to the individual innate and adaptive immunity status, sex-linked genetic variation impacts upon the rate of progression to AIDS. Elucidating the mechanisms underlying this sex-specific effect will promote the development of antiretroviral therapies with high efficacy in both sexes.

Massive visceral pentastomiasis in a long-tailed macaque - an incidental finding.

BACKGROUND: An unusual case of visceral pentastomiasis in a male adult long-tailed macaque imported from China is reported. METHODS: The monkey was part of a toxicologic study. A massive accumulation of C-shaped parasites in various visceral organs was found post-mortem. RESULTS: Based on the morphology of the nymphs, pentastomiasis was diagnosed etiopathologically. The pentastome genus and species was identified as Armillifer agkistrodontis by PCR and respective sequencing. CONCLUSION: Molecular diagnostic methods are necessary tools to determine the exact species involved.

A comparative analysis to study editing of small noncoding BC200- and Alu transcripts in brain of prion-inoculated rhesus monkeys (M. Mulatta).

Small retroelements (short interspersed elements, abbreviated SINEs) are abundant in vertebrate genomes. Using RNA isolated from rhesus monkey cerebellum and buffy coat, reverse-transcription polymerase chain reaction (RT PCR) was applied to clone cDNA of BC200 and Alu RNAs. Transcripts containing Alu-SINE sequences may be subjected to extensive RNA editing by ADAR (adenosine deaminases that act on RNA) deamination. Abundance of Alu transcripts was determined with real-time RT PCR and was significantly higher than BC200 (brain cytoplasmic) in cerebellum. BC200 transcripts were absent from buffy coat cells. Availability of the rhesus genome sequence allowed the BC200 transcripts to be mapped to the specific locus on chromosome 13. Both the qualitative and quantitative characteristics of BC 200 expression argue for the BC 200 transcripts being generated by RNA polymerase III. In cerebellum, Alu transcripts often possessed base exchanges (A to G) consistent with ADAR editing and, somewhat unexpectedly, C to T exchanges consistent with APOBEC (apolipoprotein B editing complex) editing. In contrast, the BC200 transcripts, which as RNA POLIII transcripts play a role in dendritic RNA translation, appeared not to be deaminated, despite the presence of editing of Alu in the same tissue. To assess whether neuronal disease might influence editing of BC200 and Alu-SINE transcripts in cerebellum, RNA was isolated from two rhesus monkeys that were inoculated with prions from human variant Creutzfeldt-Jakob disease (vCJD). Regardless of prion-induced neurodegeneration, no BC200 RNA editing was observed, while Alu RNA continued to show both ADAR and APOBEC editing. Thus, BC200 RNAs do not appear to become accessible to editing enzymes despite infected neurons being subjected to severe stress, damage, and eventually cell death.

Gut mucosal FOXP3+ regulatory CD4+ T cells and Nonregulatory CD4+ T cells are differentially affected by simian immunodeficiency virus infection in rhesus macaques.

The gastrointestinal tract represents a major site for human and simian immunodeficiency virus (HIV and SIV) replication and CD4(+) T-cell depletion. Despite severe depletion of mucosal CD4(+) T cells, FOXP3(+) regulatory CD4(+) T cells (T(reg)) are highly increased in the gut mucosa of chronically HIV-infected individuals and may contribute to HIV pathogenesis, either by their immunosuppressive function or as a significant target cell population for virus production. Little is known about the susceptibility of mucosal T(reg) to viral infection and the longitudinal effect of HIV/SIV infection on T(reg) dynamics. In this study, we determined the level of SIV infection in T(reg) and nonregulatory CD4(+) T cells (non-T(reg)) isolated from the colon of SIV-infected rhesus macaques. The dynamics of mucosal T(reg) and alterations in the mucosal CD4(+) T-cell pool were examined longitudinally. Our findings indicate that mucosal T(reg) were less susceptible to productive SIV infection than non-T(reg) and thus were selectively spared from SIV-mediated cell death. In addition to improved survival, local expansion of T(reg) by SIV-induced proliferation of the mucosal CD4(+) T-cell pool facilitated the accumulation of mucosal T(reg) during the course of infection. High frequency of mucosal T(reg) in chronic SIV infection was strongly related to a reduction of perforin-expressing cells. In conclusion, this study suggests that mucosal T(reg) are less affected by productive SIV infection than non-T(reg) and therefore spared from depletion. Although SIV production is limited in mucosal T(reg), T(reg) accumulation may indirectly contribute to viral persistence by suppressing antiviral immune responses.

Fatal Balamuthia mandrillaris infection in a gorilla - first case of balamuthiasis in Germany.

BACKGROUND: A 12-year-old female western lowland gorilla died in a zoological garden in Germany after exhibiting general neurological signs. METHODS: Balamuthia mandrillaris was identified as causative agent by indirect immunofluorescent staining of brain sections and confirmed by PCR and respective sequencing. RESULTS: The animal suffered from a chronic progressive necrotizing amebic meningoencephalitis. CONCLUSION: This is the first case of Balamuthia amebic encephalitis in Germany.