Heterotopic microvascular growth plate transplantation of the proximal fibula: an experimental canine model

The reconstructive potential of microvascular transplantation of skeletal growth plates was investigated through heterotopic transfers. The distal radius was resected in two series of puppies of a known large breed and substituted with a microsurgically revascularized transplant from the proximal fibula. Evaluation was conducted through serial roentgenograms, goniometric registration of joint mobility, volume measurements, histology, and fluorescent bone labeling. In the first series, development of neuropathic-like destruction of the weight-bearing graft ensued in the majority of the animals. In the second series, prolonged protection from weight bearing inhibited this destruction and resulted in hypertrophy of the revascularized epiphyseal end of the transplant but clearly reduced longitudinal growth, with only one transplant exhibiting longitudinal growth that exceeded 50 percent of the value for the control. This experiment demonstrates that skeletal growth plates possess a capacity for hypertrophy under the influence of increased loads. Whether this adaptability is sufficient to allow microvascular transplantation of growth plates to become a clinically useful procedure in children remains unclear. Further laboratory investigations are mandatory prior to clinical application of microvascular transfers of epiphyseal growth plates.     

Adoptive immunotherapy of murine cytomegalovirus adrenalitis in the immunocompromised host: CD4-helper-independent antiviral function of CD8-positive memory T lymphocytes derived from latently infected donors.

The ability of memory T lymphocytes derived from latently infected mice to control murine cytomegalovirus disease in the immunocompromised host was studied by adoptive transfer experiments. At a stage of pathogenesis when virus had already colonized target tissues, a therapeutic antiviral function could be ascribed to the CD8+ subset. This in vivo function was not restricted to sites in which intravenously infused lymphocytes usually are trapped or home in, such as the lungs or the spleen, respectively, but was also evident in the adrenal glands, a site to which antiviral effector cells have to specifically migrate. Specific infiltration of adrenal gland cortical tissue by donor-derived CD8+ memory T lymphocytes was demonstrated. CD4+ memory T lymphocytes had no antiviral effect by themselves and also were not required for the function of the CD8+ effector cells in this short-term immunotherapy model. These findings should help settle the debate about which subset of T lymphocytes comprises the effector cells that can directly control cytomegalovirus infection in the murine model system.     

Vascularized bone allografts: in vitro assessment of cell-mediated and humoral responses

The immunologic consequences of transplantation of vascularized bone allografts have not been previously characterized. In this study, knee allografts, both vascularized and nonvascularized, were transplanted from Lewis rats to Brown Norway rats across a strong histocompatibility barrier. A total of 66 transplants and 8 control animals were evaluated. The vascularized knee grafts consisted of 1 cm of proximal tibia and distal femur with a minimal muscular cuff isolated on the femoral vessels, and these were transplanted to a heterotopic, subcutaneous position on the abdominal wall of the recipient rat. Nonvascularized allografts (identical but without anastomoses) were transplanted for comparison. The cell-mediated response was measured by lymphocytotoxicity assay, and the humoral response was measured by cytotoxic antibody assay, both employing 51Cr-labeled target cells. The timing and intensity of the immune response differed according to the type of graft. The vascularized bone allografts generated significant cell-mediated and humoral responses as early as 5 days posttransplant. A significant humoral response in nonvascularized bone allografts was not apparent until day 14, while cell-mediated response in these grafts was variable. These findings were correlated with the histologic appearance of the grafted tissue. Cyclosporine, which was administered to one group of vascularized bone allografts, resulted in the suppression of both types of immune responses. The histologic appearance of this group resembled that of isografts transplanted as controls. The clinical application of vascularized bone allografts may offer significant advantages over nonvascularized allografts in the reconstruction of massive bone defects. Complications such as nonunion, fracture, and collapse of articular segments seen in nonvascularized allograft transplantation may be avoided by preservation of the blood supply to the graft. Characterization of the immune response to vascularized bone allografts may subsequently allow the manipulation of the host and/or graft tissue and promote graft incorporation.     

Evaluation of Diverse Antisera, Conjugates, and Support Media for Detecting Bradyrhizobium japonicum by Indirect Enzyme-Linked Immunosorbent Assay

We evaluated three antisera and four enzyme conjugates for the detection of Bradyrhizobium japonicum by an indirect enzyme-linked immunosorbent assay in microtiter plates. Nitrocellulose membrane sheets were then evaluated as an alternative support medium by using some combinations. Partially purified immunoglobulin G (IgG) or unpurified antisera to strain USDA 110 raised in rabbits, goats, or sheep was reacted in microtiter plates with alkaline phosphatase conjugated to protein A, goat anti-rabbit (GAR), sheep anti-rabbit (SAR), or rabbit anti-goat (RAG) IgG. Cultures or nodules containing homologous rhizobia were detected with equal sensitivity when protein A, GAR, or SAR was reacted with 5 μg of protein IgG per ml or a 1:800 titer of antisera from rabbits, but not goats or sheep. RAG reacted with IgG or antisera from goats or sheep. The detection limit was 2 × 10⁵ rhizobia per well. Rhizobia were spotted on nitrocellulose sheets as an alternative support medium, followed by soaking in 5 μg of protein per ml as IgG and 1:4,000 dilutions of protein A or GAR conjugate. Rhizobia in serogroup 110 were detected with the dye combination Nitro Blue Tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP), and rhizobia in serogroup 122 were detected with fast red-naphthol phosphate (FR-NP). At the conclusion of the 5-h assay, purple (NBT-BCIP) or red (FR-NP) spots were visible in positive reactions. The sensitivity of detection was about 1,000 rhizobial cells or 3 μg of nodules tissue.     

Untersuchcungen zum Verhalten von Cryptococcus Neoformans in Erde

Zusammenfassung Innerhalb eines nachgewiesenermaßen mitCryptococcus neoformans (Cr. n.) infizierten Stadtgebietes wurden 170 Erdproben aufCr. n. untersucht. Während aus 6 von 50 Erdproben von Lokalisationen mit TaubenbesiedelungCr. n. nachgewiesen wurde, gelang dies bei keiner von 120 Proben ohne Taubenbesiedelung.Diese Engebnisse werden so gedeutet, daß zumindest unter den hiesigen BedingungenCr. n. als primärer Erdbodensaprophyt praktisch nicht vorkommt. Die Besiedelung von Taubenkot mitCr. n. geschieht vielmehr bereits im Darmkanalspontan infizierter Tauben, oder durch Kontakt mit infiziertem Kot. Hierbei gelangensekundär auch Kryptokokken in den Erdboden.Für künftige epidemiologische Untersuchungen wird eine Nachweismethodik empfohlen, die den Direktausstrich des in phys.-Kochsalzlösung suspendierten Untersuchungsmaterials auf Negersaat-Kreatinin-Antibiotika-Diphenylagar, seine Anreicherung in diphenylhaltiger (0,002 %) Bierwürze und den Tierversuch an der weißen Maus umfaßt. Mit dieser Methodik ist beim Vorhandensein von mindestens 10Cr. n. -Zellen in 1 g untersuchtem Ausgangsmaterial der Nachweis des Erregers zu erwarten.Halbquantitative Antrocknungsversuche vonCr. n. (ein stark und ein nahezu unbekapselter Stamm, zwei verschiedene Antrocknungsmethoden) an sterilen Seesand zeigten, daß selbst die unter direkter Einwirkung von Sonnenlicht angetrockneten Kryptokokken weitgehend überlebten.Die Direktkultur war dem Anreicherungsverfahren nahezu gleichwertig, während sich der Tierversuch im Vergleich dazu als weit weniger leistungsfähig erwies. Unter Hinweis auf frühere Untersuchungen wird betont, daß auf den Tierversuch dennoch nicht verzichtet werden sollte.

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Within the area of a city, well known to be infected withCryptococcus neoformans, 170 soil samples have been for this fungus investigated. While six out of 50 soil samples coming from pigeon locations were positive for the fungus, none of the other 120 soil samples became positive from areas without pigeon locations. For future epidemiologic investigations a special method is suggested. The direct culture of the material was almost equivalent with the method of enrichment, while the animal experiment in comparison was less efficient. It is very probable, thatCryptococcus neoformans is no primary germ of the soil. Soil becomes secondarily contaminated by pigeons and their excrements.

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Die Untersuchungen wurden dankenswerterweise durch Mittel des Bundesministers für Ernährung, Landwirtschaft und Forsten gefördert.     

Characterization of radiolabeled agonist binding to beta-adrenergic receptors in mammalian tissues

Recent evidence suggests that the molecular interactions of agonists with beta-adrenergic receptors differ from those of antagonists. Most of this evidence has come from studies of agonist inhibition of radiolabeled antagonist binding. We have examined agonist binding directly in rat lung membranes using radiolabeled hydroxybenzylisoproterenol (3H-HBI). Specific binding of 3H-HBI was stereoselective and was inhibited by catecholamines with a potency order characteristic of beta 2-adrenergic receptors. Gpp(NH)p increased the rates of association and dissociation of 3H-HBI from the receptor. In the absence of Gpp(NH)p, Scatchard plots were curvilinear suggesting a complex interaction of the agonist with the receptor. The total number of 3H-HBI binding sites was similar to that of 125I-IHYP binding sites. In the presence of increasing concentrations of Gpp(NH)p, the affinity of 3H-HBI was decreased and Scatchard plots became linear. Sodium chloride mimicked the effect of Gpp(NH)p in lowering the affinity of the receptor for 3H-HBI. Magnesium chloride had the opposite effect in that it promoted high affinity binding. The effect of sodium chloride was largely overcome by the presence of magnesium chloride.     

Deactivation of catalase by hydrogen peroxide

When a mechanism is used to determine possible deactivation kinetics, a certain consistency with the kinetics of the main reaction is found. This result is examined in the light of experimental evidence obtained with bovine liver and Aspergillus catalases.     

Studies on the deactivation of immobilized urease

The results of experiments in a fixed-bed reactor and a CSTR containing urease immobilized on a nonporous support and conducted in the absence of diffusional limitations are reported. Kinetic parameters were established by separate batch experiments. The key observation was that the product ammonia attacked the free form of the enzyme and thereby illustrates the importance of mechanism in determining deactivation kinetics.