Eotaxin/CCL11 expression by infiltrating macrophages in rat heart transplants during ongoing acute rejection

Eotaxin/CCL11 chemokine is expressed in different organs, including the heart, but its precise cellular origin in the heart is unknown. Eotaxin is associated with Th2-like responses and exerts its chemotactic effect through the chemokine receptor-3 (CCR3), which is also expressed on mast cells (MC). The aim of our study was to find the cellular origin of eotaxin in the heart, and to assess whether expression is changing during ongoing acute heart transplant rejection, indicating a correlation with mast cell infiltration which we observed in a previous study.In a model of ongoing acute heart transplant rejection in the rat, we found eotaxin mRNA expression within infiltrating macrophages, but not in mast cells, by in situ-hybridization. A five-fold increase in eotaxin protein in rat heart transplants during ongoing acute rejection was measured on day 28 after transplantation, compared to native and isogeneic control hearts. Eotaxin concentrations in donor hearts on day 28 after transplantation were significantly higher compared to recipient hearts, corroborating an origin of eotaxin from cells within the heart, and not from the blood.The quantitative comparison of eotaxin mRNA expression between native hearts, isografts, and allografts, respectively, revealed no statistically significant difference after transplantation, probably due to an overall increase in the housekeeping gene's 18S rRNA during rejection. Quantitative RT-PCR showed an increase in mRNA expression of CCR3, the receptor for eotaxin, during ongoing acute rejection of rat heart allografts.Although a correlation between increasing eotaxin expression by macrophages and mast cell infiltration is suggestive, functional studies will elucidate the role of eotaxin in the process of ongoing acute heart transplant rejection.     

Trypanosoma cruzi myocardial infection reactivation presenting as complete atrioventricular block in a Chagas'' heart transplant recipient

A 56-year-old man underwent orthotopic heart transplantation because of end-stage Chagas' cardiomyopathy. One hundred and ten days following heart transplantation, an electrocardiogram tracing showed complete atrioventricular block, which was treated with temporary transvenous pacemaker insertion. An underlying endomyocardial biopsy was graded 3A. The patient was treated with pulse steroid therapy. One week later, the patient died of multiorgan failure secondary to septicemia. A careful review of the endomyocardial biopsy showed nests of parasites in the myocardial tissue accompanied by mononuclear cell infiltrate similar to that found in acute graft rejection. Thus, complete atrioventricular block may be another clinical manifestation of Trypanosoma cruzi infection reactivation in Chagas' heart transplant recipients.     

Human fetal tissues grafted to rodent hosts: structural and functional observations of brain,adrenal and heart tissues in oculo

Summary The potential for growth and development of human tissue grafts was explored by transplantation to the anterior chamber of the eye of rats and mice. Tissues were obtained from therapeutic abortions, performed in the eighth to twelfth week of gestation, using a slight modification of routine vacuum aspirations. Recipients were either adult rats immunosuppressed with cyclosporin A and protected with antibiotics, or nude immunodeficient Balb C mice. Catecholamine-rich tissues such as chromaffin cells from the adrenal medulla, sympathetic ganglia, central dopamine neuroblasts from the substantia nigra, and noradrenaline neuroblasts from the locus coeruleus all survived grafting, and in many cases formed nerve fibers that invaded the host iris. Similarly, central serotonin neurons from developing raphe nuclei grafts were able to innervate host irides. Human fetal cerebellar and cerebral cortical transplants continued their development in rat host eyes. Extracellular recordings from such cerebellar and cortical grafts revealed spontaneously active cells with immature action potential waveforms. Spinal cord grafts also survived and contained substance P-immunoreactive neurons. Dorsal root ganglia were able to form nerve fibers invading the host iris, as evidenced by neurofilament immunohistochemistry. Heart tissue survived and manifested spontaneous rhythmic contractions in oculo. Both human cortex cerebri and heart tissue grafts became innervated by sympathetic adrenergic nerve fibers from the rat host iris. Thus both graft-to-host and host-to-graft neuronal connections may be established between man and rat. Taken together, these data suggest that transplantation of human fetal nervous tissues to the anterior chamber of immunosuppressed or immunodeficient rodent hosts yields a unique model system for studies of human brain development, developmental disturbances, connectivity, and the action of drugs.     

Pre- and posttransplantation allosensitization in heart allograft recipients: major impact of de novo alloantibody production on allograft survival

The involvement of humoral response in allograft rejection has been suggested by both immunologic and histochemistry studies. In the present study, we explored the role of alloantibodies in a large cohort of heart allograft recipients followed for 15 years. Sequential samples of sera were obtained from 950 recipients of heart allografts before and after transplantation at the time when protocol endomyocardial biopsies were performed. The presence of anti-human leukocyte antigen (HLA) antibodies was investigated using complement mediated cytotoxicity and solid phase assay (SPA). Our data reveal an inverse correlation between the development of alloantibodies after transplantation and heart allograft survival. The 15-year graft survival was highest in patients who never developed alloantibodies (70%) or who displayed them only before transplantation (71%); graft survival in recipients who showed antibodies both before and after transplantation (56%), or only after transplantation (47%), was lower. The deleterious effect of antibodies on graft survival started 8 years after transplantation, suggesting that the production of de novo antibodies may have been triggered by some later event. We found that patients with de novo antibodies appearing more than 1 year after transplantation had the poorest survival. Furthermore, the development of de novo antibodies was preceded in 76% of these patients by cellular rejection grade 3 or higher, according to the International Society for Heart Transplantation (ISHT) grading criteria. Development of antibody-mediated rejection (AMR) had a significant negative impact on graft survival (16% in AMR(+) vs 63% in AMR(-) patients, p = 0.0008). Of the 23 patients with AMR, 21 displayed cytotoxic donor-specific antibodies (DSA) at the time of diagnosis, and in 18 of these cases SPA showed that they were directed against the donors' HLA. The data demonstrate that the detection of alloantibodies permits a better definition of AMR in heart allograft recipients. Identification of patients at risk for developing AMR is of great importance for early treatment of rejection episodes.     

HMGB1 blockade differentially impacts pulmonary inflammation and defense responses in poly(I:C)/LPS-exposed heart transplant mice

A large number of recipients are in a compromised immune defense condition because of the routine application of immunosuppressive regimens after heart transplantation. Our previous work demonstrated that blockade of high-mobility group box 1 (HMGB1) prolongs the graft survival. Whether and how HMGB1 blockade impacts respiratory responses against pathogen-like challenge in organ transplant recipients when it improves cardiac graft are not elucidated. At the present study, after abdominal heterotopic heart transplantation, the recipient mice were treated with HMGB1 mAb, and then challenged with poly(I:C) or LPS intratracheally on day 7 post transplantation. We found that the level of bronchoalveolar lavage (BAL) HMGB1 was elevated after heart transplantation, and aggravated responses to respiratory tract poly(I:C)/LPS challenge were observed. HMGB1 neutralizing mAb treatment in poly(I:C)-challenged recipient mice alleviated pulmonary histopathological changes, neutrophil infiltration and inflammatory cytokine release, but unaffected the level of IFN-β, the distribution of CD11b⁺CD27⁺/CD11b⁺CD27⁻ NK cell subsets, and CD8⁺ T cell responses. In LPS-exposed recipient mice, HMGB1 mAb treatment ameliorated pulmonary inflammatory damage and enhanced the phagocytosis of phagocytic cells. Thus, this study may establish a basis for the application of HMGB1 blockade to improve the outcomes of heart transplant recipients because HMGB1 inhibition ameliorates pulmonary inflammation, but maintains defense-associated responses.     

Current status of left ventricular assist devices: the role in bridge to heart transplantation and future perspectives

Increasing numbers of patients with end-stage heart failure are awaiting heart transplantation worldwide. Left ventricular assist systems have been applied to these patients with increasing frequency and support duration. This situation calls for more reliably applied long-term support devices and is also stimulating the investigation of destination therapy or bridge to recovery use of the device. This is particularly relevant in countries where organ shortages are crucial. However, reports in this area are still limited, and there exist several problems to be solved before wider application of these strategies is possible in clinical practice. We review currently available devices with special reference to their respective roles and limitations quoting our own experiences over the past several years, and discuss future perspectives of ventricular assist devices.     

Alterations of heart development in Xenopus laevis by galactoside-binding lectin or its sugar hapten inhibitor

The early heart anlagen of Xenopus laevis embryos were exposed to purified embryonic galactoside-binding lectin or its potent hapten inhibitor thiodigalactoside (TDG). Heart development was then studied using a variety of microscopical techniques. Conotruncal morphology and positioning with respect to the ventricle are altered in treated animals. In 34% of animals treated with lectin and 35% treated with TDG, the conotruncus leaves the ventricle from an abnormal location. Lectin or TDG treatments are also correlated with altered conotruncal shape, with the conotruncal regions showing greater radii of curvature compared to controls. Conotruncal myocyte differentiation is altered by the test treatments, with lack of development of organized myofibrillar arrays. Conotruncal cushion development is also affected. Changes occur in the shape and size of the primary conotruncal cushion, and alterations of outflow tract septation develop. Less maturation of ventricular myocytes is also observed in test animals. The results suggest that galactose-lectin interactions are important in heart development.     

Domestic and foreign trends in the prevalence of heart failure and the necessity of next-generation artificial hearts: a survey by the Working Group on Establishment of Assessment Guidelines for Next-Generation Artificial Heart Systems

A series of guidelines for development and assessment of next-generation medical devices has been drafted under an interagency collaborative project by the Ministry of Health, Labor and Welfare and the Ministry of Economy, Trade and Industry. The working group for assessment guidelines of next-generation artificial hearts reviewed the trend in the prevalence of heart failure and examined the potential usefulness of such devices in Japan and in other countries as a fundamental part of the process of establishing appropriate guidelines. At present, more than 23 million people suffer from heart failure in developed countries, including Japan. Although Japan currently has the lowest mortality from heart failure among those countries, the number of patients is gradually increasing as our lifestyle becomes more Westernized; the associated medical expenses are rapidly growing. The number of heart transplantations, however, is limited due to the overwhelming shortage of donor hearts, not only in Japan but worldwide. Meanwhile, clinical studies and surveys have revealed that the major causes of death in patients undergoing long-term use of ventricular assist devices (VADs) were infection, thrombosis, and mechanical failure, all of which are typical of VADs. It is therefore of urgent and universal necessity to develop next-generation artificial hearts that have excellent durability to provide at least 2 years of event-free operation with a superior quality of life and that can be used for destination therapy to save patients with irreversible heart failure. It is also very important to ensure that an environment that facilitates the development, testing, and approval evaluation processes of next-generation artificial hearts be established as soon as possible. Working Group on Establishment of Assessment Guidelines for Active Implantable Medical Devices: Next-Generation Artificial Heart System, Inter-agency Committee for the Guideline Program, the Ministry of Health, Labour and Welfare of Japan and the Ministry of Economy, Trade and Industry of Japan, Tokyo, Japan     

Monodelphis domestica (grey short-tailed opossum): an accessible model for studies of early neocortical development

Summary The endocardium ultrastructure of 13 embryonic day old hamsters was examined, especially in relationship with the atrial myocytes. The endothelial morphology was described, including the junctional attachments and their relationships with subjacent atrial myocytes. Characteristic atrial myocytes organelles were identified: myofibrils, atrial granules, lipidic inclusions, and polysomes. Immunogold labeling demonstrated that atrial natriuretic factor (ANF)-containing granules were already present in the differentiating cardiomyocytes, even before the myofibrils were completely organized. At this stage of development, while the endothelium was a narrow barrier between the blood and the cardiomyocytes, it displayed fenestrations, but also epithelial discontinuities. In addition it also contains immunoreactive-ANF products. In light of the current knowledge about ANF processing it was proposed that the endocardium lining could be an obligated passageway for transport or activating proANF into ANF before its release into the blood stream. In addition the endocardial gaps could suggest that, until about 13 to 14 days of fetal development, heart atrial tissue could be more susceptible to the effects of pathogenetic compounds than in a later state of development.     

Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation (SBMT).

MRL-lpr/lpr mice spontaneously develop a severe autoimmune syndrome, characterized by massive generalized lymphadenopathy, arthritis, arteritis, dermatitis and immune complex-mediated glomerulonephritis. Bone marrow transplantation (BMT) from MHC-matched systemic lupus erythematosus (SLE)-resistant donors to susceptible recipients has proved effective in correcting autoimmune manifestations in autoimmune-prone mice. We investigated the effect of syngeneic BMT from MRL/lpr (donor) to immunocompromised MRL/lpr (recipient), after purging the bone marrow inoculum with MoAbs against mature T cells (anti-Thy 1.2). All the untreated mice developed lymphadenopathy and by the age of 36 weeks five of the eight were dead; in contrast, all the mice which underwent syngeneic BMT following acute immunosuppression with total body irradiation (900 cGy) (TBI) remained disease-free. In an additional experiment, it was found that conditioning with cyclophosphamide (CY) before BMT was more effective than TBI in inhibiting delayed-onset autoimmune manifestations (mean survival 350 days in the CY group and 305 days in the TBI group, versus 197 days in untreated controls). Under both immunosuppressive regimens T cell-depleted bone marrow grafts produced far better results than did unmanipulated BMT. Following syngeneic BMT the incidence of proteinuria and the level of serum anti-DNA (dd) antibodies were significantly reduced, compared with that of the age-matched untreated controls. CY was more effective than TBI in reducing the anti-DNA titres. Likewise, T depletion of bone marrow inocula before BMT induced a more drastic drop in autoantibodies, following both CY and TBI conditioning protocols. After syngeneic BMT (either CY or TBI) no signs of lymphadenopathy were observed even at an advanced age. Upon histopathological examination, the BMT-treated mice displayed normal glomeruli with occasional minimal signs of glomerulonephritis. Syngeneic T cell-depleted BMT following acute cytoreduction of anti-self immune lymphocytes may represent a new therapeutic approach for drug-resistant autoimmune diseases.     

The effect of O2 and CO2 on the dive behavior and heart rate of lesser scaup ducks (Aythya affinis): quantification of the critical that initiates a diving bradycardia

Lesser scaup ducks were trained to dive for short and long durations following exposure to various gas concentrations to determine the influence of oxygen (O₂) and carbon dioxide (CO₂) on diving behavior and heart rate. Compared with normoxia, hyperoxia (50% O₂) significantly increased the duration of long dives, whereas severe hypoxia (9% O₂) significantly decreased the duration of both short and long dives. Hypercapnia (5% CO₂) had no effect on dive duration. Surface intervals were not significantly altered by the oxygen treatments, but significantly increased following CO₂ exposure. Heart rate during diving was unaffected by hyperoxia and hypercapnia, but gradually declined in long dives after severe hypoxia. Thus, our results suggest that during the majority of dives, O₂ and CO₂ levels in lesser scaup ducks are managed through changes in diving behavior without any major cardiovascular adjustments, but below a threshold Pa_O2, a bradycardia is evoked to conserve the remaining oxygen for hypoxia sensitive tissues. A model of oxygen store utilization during voluntary diving was developed to estimate the critical Pa_O2 below which bradycardia is initiated (≈26 mmHg) and predicted that this critical Pa_O2 would be reached 19 s into a dive after exposure to severe hypoxia, which corresponded exactly with the time of initiation of bradycardia in the severe hypoxia trials.     

Ultrastructural study of the sinus venosus in embryos of the dogfish ( Scyliorhinus canicula )

 An ultrastructural study of the development of the sinus venosus has been carried out on seven embryos of the dogfish (Scyliorhinus canicula L.) between 10.5 and 69 mm of total length (TL). The sinus venosus appears at the end of the looping process of the cardiac tube, namely in the 10.5 mm embryo, when the heart reaches its adult tetracameral S-form. The endocardium of the smallest embryo is constituted of a single layer of cubic cells. In larger embryo, these cells progressively acquire a squamous appearance as well as electron-dense cytoplasmic inclusions. The subendocardium is progressively populated by ganglion cells, Schwann cells and bundles of amyelinic fibers that can first be recognised in the embryo of 34 mm TL. Some subendocardial mesenchymal cells located in earlier embryos close to the entrance of the ducts of Cuvier might be their ectomesenchymal progenitors. The myocardium is initially constituted of a single layer of cubic cells. In the embryos of 19, 27 and 34 mm TL, the myocardium becomes multilayered, and the myocardiocytes develop myofibrils randomly arranged throughout the sarcoplasm. In later embryos, the myocardiocytes are innervated and arranged in oval bundles surrounded by a basal lamina. The epicardium covers the sinus venosus by the retrograde migration of the epithelium already established around the atrioventricular groove and, in a lesser degree, by the adhesion of mesothelial cells that are floating free in the pericardial cavity. This process has finished in the embryo of 34 mm TL. The differentiation of the sinus venosus (including the endocardial and myocardial differentiation as well as the epicardial covering) progresses in an anteroventral-posterodorsal direction. Accepted: 12 June 1998     

Absence of formation of benzo[a]pyrene/DNA adducts in the cuttlefish (sepia officinalis,mollusca: Cephalopoda)

Benzo[a]pyrene (B[a]P) injected intramuscularly into the base of the arms of cuttlefish was released continuously from the injection site and removed from the organism. Only a portion of the compound accumulated in the body. Twenty-four hr after its injection, 75% of B[a]P applied in olive oil was removed from the cuttlefish, and 1.2% was found in the body outside the head, the site of injection. If the carcinogen was dissolved in dimethylformamide, the removal of B[a]P was slower, so that only 18% of the injected B[a]P was removed from the organism and 0.36% accumulated in the body outside the head 24 hr after injection. The high level of B[a]P in gills and hemolymph 4 hr after injection and the kinetics of the decrease of its concentration with time indicate that these two organs could be involved in the excretion of B[a]P from the body. The B[a]P/DNA adducts characteristic for vertebrates could not be demonstrated in gills, skin, brain, hepatopancreas, and lymphocytes of the cuttlefish 24 hr after injection of B[a]P. The dose of the carcinogen injected into the cuttlefish was 2—4 times higher than the dose resulting in the formation of a high level of B[a]P/DNA adducts in the vertebrates. A different metabolism of B[a]P in the tissue of cephalopods, compared to vertebrates, could be less favorable to the process leading to malignant transformation and could explain the absence from the literature of reports of tumors in cephalopods. © 1994 Wiley-Liss, Inc.     

Concomitant heart rate and corneoretinal potential conditioning in the rabbit (Oryctolagus cuniculus): effects of caudate lesions

Rabbits received either bilateral, unilateral, or sham caudate lesions and were subjected to Pavlovian conditioning training. Corneoretinal potential (CRP), electromyographic (EMG), and heart rate (HR) CRs were assessed. Lesions which destroyed the anteromedial two thirds of the head of the caudate nucleus greatly impaired the acquisition of the CRP response during both simple and differential Pavlovian conditioning. However, the magnitude of the HR CR and the HR discrimination were unaffected by these lesions. Measures of free field activity, electromyographic activity, and CRP thresholds to shock revealed no evidence of a motor or sensory deficit in the lesioned animals. These data thus demonstrate a deficit in a specific learned somatomotor response but no impairment in the autonomic changes which usually accompany somatomotor conditioning.     

Role of the mixed lymphocyte culture (MLC) reaction in marrow donor selection: Matching for transplants from related haploidentical donors

Abstract: The utility of the MLC assay as a test of HLA-D region matching and predictor of acute graft-versus-host disease (GvHD) was evaluated in 157 patients receiving marrow grafts from HLA-A, B identical related haploidentical donors. All donors and recipients were tested by HLA-DR serology, by Dw phenotyping with homozygous typing cells (HTC) and by standard MLC. Ninety-nine of the donor-recipient pairs were mismatched for a serologically defined HLA-DR antigen while 109 pairs were mismatched for the HLA-D region by HTC typing. Donor anti-recipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from -4% to 100%, with a median of 25%. A comparison of reactivity in MLC with presence or absence of matching by Dw phenotyping, however, showed a significant overlap in the distribution of RRs from HLA-Dw matched versus Dw mismatched pairs, suggesting that the MLC was not a reliable predictor of HLA-Dw matching. Using an optimally-defined cutoff of 3% RR, the MLC was correlated with risk of developing clinically significant grades II–IV acute GvHD (p = 0.03) but not with risk of developing severe grades III-IV GvHD (p = 0.18). In contrast, matching by Dw phenotype was a significant predictor of GvHD, with Dw-compatible transplant recipients less likely to develop either grades II-IV (p = 0.004) or III-IV (p = 0.036) GvHD than Dw-incompatible transplant recipients. Overall, these results underscore the difficulty in using the MLC to measure HLA-D region compatibility and predict the risk of severe graft-versus-host disease among patients receiving related haploidentical marrow grafts. HLA-D (HTC) typing results correlate primarily with DRB compatibility, and with the advent of DRB1 allele matching by sequence-specific oligonucleotide probes (SSOP) or by direct sequencing, the precision in donor matching achievable with these methods is far greater than with either HLA-D typing or direct MLC testing.     

Processing of alkylcobalamins in mammalian cells: A role for the MMACHC (cblC) gene product

The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B₁₂). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5′-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using [⁵⁷Co]-AdoCbl and MeCbl analogs consisting of [⁵⁷Co]-labeled straight-chain alkylcobalamins ranging from C2 (ethylcobalamin) to C6 (hexylcobalamin). [⁵⁷Co]-AdoCbl was converted to [⁵⁷Co]-MeCbl by cultured bovine aortic endothelial cells, suggesting that a dealkylation process likely involving the cblC protein removed the 5′-deoxyadenosyl alkyl group. Surprisingly, all of the straight-chain alkylcobalamins served as substrates for the biosynthesis of both AdoCbl and MeCbl. Dealkylation was then assessed in normal skin fibroblasts and fibroblasts derived from three patients with mutations in the MMACHC gene. While normal skin fibroblasts readily converted [⁵⁷Co]-propylcobalamin to [⁵⁷Co]-AdoCbl and [⁵⁷Co]-MeCbl, there was little or no conversion in cblC mutant fibroblasts. These studies suggest that the CblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation) in mammalian cells.     

Accumulation of rare sex chromosome rearrangements in the African pygmy mouse, Mus (Nannomys) minutoides: a whole-arm reciprocal translocation (WART) involving an X-autosome fusion

Although sex chromosomes are generally the most conserved elements of the mammalian karyotype, those of African pygmy mice show three extraordinary deviations from the norm: (a) asynaptic sex chromosomes, (b) multiple sex–autosome fusions, and (c) modifications of sex determination in some populations/species. In this study we identified, in two sex-reversed females of Mus (Nannomys) minutoides, a fourth rare sex chromosome change: a spontaneous whole-arm reciprocal translocation (WART) between an autosomal Robertsonian pair Rb(13.16) and the sex–autosome fusion Rb(X.1). This represents one of the very few reported cases of WARTs in natura within mammals, and is the first one to involve sex chromosomes. Hence, this finding offers new insights into the mechanisms of chromosomal differentiation in African pygmy mice, as WARTs may have contributed to the extensive diversity not only of autosomal Robertsonian fusions, but also of sex–autosome translocations. More widely, these results provide additional support to previous studies on the house mouse and the common shrew which indirectly inferred the role of WARTs in their karyotypic evolution, and may even help to understand how the fascinating 10 sex chromosome chain of the platypus might have evolved. This accumulation of rare sex chromosome changes in single specimens is, to our knowledge, exceptional among mammals.