Christian Hasse

Parathyroid Xenotransplantation without Immunosuppression in Experimental Hypoparathyroidism: Long-term In Vivo Function following Microencapsulation with a Clinically Suitable Alginate

Permanent hypoparthyroidism is one of the most difficult of all endocrine disorders to treat medically. Because this deficiency syndrome rarely is a life-threatening condition, systemic immunosuppression for recipients of transgenic transplants is not justified. An alternative would be protecting the tissue to be transplanted from the immunologic response (immunoisolation) by coating it with a semipermeable membrane- microencapsulation. Unfortunately, prior to the first clinical use, further analysis of the coating substance (alginate) demonstrated that it has potential cancerogenic properties. Using a purified amitogenic alginate suitable for clinical use, we accomplished allotransplantation in a long-term animal model and reported the first clinical cases without postoperative immunosuppression recently. In view of the potential clinical use, we investigated the ability of the microencapsulation technology with the novel amitogenic alginate in experimental hypoparathyroidism (80 parathyroidectomized rats) to enable transgenic transplantation across the highest immunologic barrier (xenotransplantation: human to rat) to ensure intact transplant function without immunosuppression. In a controlled, long-term animal study, the effect of microencapsulation on xenotransplanted human parathyroid tissue was evaluated over a period of 30 weeks (microencapsulated and naked hyperplastic parathyroid tissue, respectively). Functionally, human parathyroid tissue was able to replace that of rats. More than 6 months after xenotransplantation 32 of 40 animals that had received microencapsulated transplants were normocalcemic. In contrast, serum calcium concentrations dropped to postparathyroidectomy levels within 3 weeks in the animals that had received naked tissue only. Correspondingly, normocalcemic animals showed vital parathyroid tissue inside the microcapsules, which were surrounded by a small rim of fibroblasts. When combining microencapsulation with an improved tissue culture method, xenotransplantation of human parathyroid tissue and maintenance of its physiologic function is reproducibly achieved over the highest transplantation barrier. Using the amitogenic alginate may be a crucial step toward the first clinical use of this technique for parathyroid xenotransplantation without immunosuppression.

Quality of Life and Patient Satisfaction after Reoperation for Primary Hyperparathyroidism: Analysis of Long-term Results

orbidity after reoperation for persistent or recurrent primary hyperparathyroidism (pHPT) is higher than after primary surgery. According to our experience, there is a contrast between postoperative normalization of laboratory parameters and the quality of life/patient satisfaction after reoperation. Therefore the aim of the study was to analyze the outcomes of reoperations in comparison to primary surgery. We evaluated the patients’ reported quality of life using the SF-36 (an accepted health status assessment tool) and complete prospectively documented perioperative and follow-up data including postoperative complications. Additionally, we searched for reasons why primary surgical intervention did not succeed. In a prospective cohort study the perioperative data of 653 consecutive patients with pHPT, including 75 reoperated patients (11.5%) who underwent parathyroidectomy between 1987 and 1999, were evaluated by uni- and multivariate analysis. At a median 78 months (6–156 months) postoperatively, all patients underwent a planned follow-up that included the SF-36, physical examination, and laboratory investigations. A total of 51 reoperated patients were available for follow-up. Postoperative alleviation of symptoms or being symptom-free was reported by 70.6%. Patients after reoperation had lower SF-36 scores in all health domains postoperatively than patients after a primary operation. Of the reoperated patients, 19.6% stated that after evaluating the development of their complaints they would not consent to reoperation again. Subgroup analysis showed that 80% of patients with postoperatively persistent pHPT, 60% of those who did not observe symptom alleviation, and 44% of those after sternotomy were in the group of dissatisfied patients. Surprisingly, none of the patients with more than one reoperation, only two of the five patients with permanent recurrent laryngeal nerve injury, and only one of the four patients with persistent hypoparathyroidism were dissatisfied overall. Parathyroidectomy resulted in normocalcemia in 90.2% of the reoperated patients, with an operative morbidity of 27.4% and no mortality. After an unsuccessful operation for pHPT, patients should be treated at an expert center to avoid persistent hypercalcemia. Reoperations necessitating sternotomy should be restricted to patients with severe symptoms and signs.

Amitogenic Alginates: Key to First Clinical Application of Microencapsulation Technology

Abstract. Microencapsulation refers to a technique of immunoisolation by coating single cells or tissue with a semipermeable membrane. By combining microencapsulation with a specific tissue culturing method, iso-, allo-, and xenotransplantation of parathyroid tissue has been achieved without immunosuppression in a long-term animal model. Prior to its clinical use, continued analyses of the alginate, used as a coating substance, determined its mitogenic properties. Purification of the commercially available alginate was achieved using patented electrophoretic procedures, resulting in an amitogenic alginate suitable for use in humans. However, this alginate exhibited entirely different physical properties. We have recently shown that isotransplanted parathyroid tissue remains vital and functioning in vivo over long periods of time using the novel amitogenic alginate. It is essential to document, whether the alginate is able to maintain immunoisolation. We have therefore assessed its in vivo function compared to the mitogenic alginate in a transgenic animal model. Altogether 600 parathyroid glands from 300 Lewis rats (donor animals) were excised and subjected to tissue culture. Thereafter they were allotransplanted to 30 parathyroidectomized Dark-Auita rats, microencapsulated with the amitogenic or the mitogenic alginate or naked, with 10 recipient animals in each group. Total serum calcium and parathyroid hormone levels were monitored continuously at weekly intervals for 30 weeks. After 26 weeks the transplant beds were excised and subjected to histologic examination. More than 6 months after allotransplantation 9 of 10 animals that had received amitogenic transplants, compared to 7 of 10 animals in the group with mitogenic microcapsules were normocalcemic. Animals that had received naked parathyroid tissue were hypocalcemic as soon as 2 weeks after allotransplantation. Correspondingly, normocalcemic animals showed vital parathyroid tissue inside the microcapsules, which were surrounded by a significantly smaller rim of fibroblasts when amitogenic alginate had been used. In addition to confirming physiologic long-term function, we were able to document for the first time that immunoisolation can also be achieved with the novel amitogenic alginate, which is suitable for clinical use.

First successful xenotransplantation of microencapsulated human parathyroid tissue in experimental hypoparathyroidism: long-term function without immunosuppression.

Owing to the complexity of the parathyroid hormones metabolic interactions, clinical hypoparathyroidism is one of the most difficult of all endocrine disorders to treat. Therefore, causative treatment of this disorder by transplantation of parathyroid glands is highly desirable. We have recently documented the long-term in vivo function of iso- and allotransplanted rat parathyroid tissue without systemic immunosuppression in an animal model. In view of the potential clinical use of this method, human parathyroid tissue has been microencapsulated and transplanted over the highest immunological barrier. In a controlled, long-term animal study in the parathyroidectomized rat, the effect of microencapsulation on xenotransplanted human parathyroid tissue was evaluated over 30 weeks (native and microencapsulated parathyroid tissue = 40 rats respectively). Functionally, human parathyroid tissue was able to replace that of the rat. All animals that had received microencapsulated parathyroid tissue were normocalcemic for 16 weeks; 27/40 at the end of the study. In contrast, serum calcium concentrations dropped to post-parathyroidectomy levels within 4 weeks in those animals that had received native tissue only. Histologic evaluation of the explanted, functionally successful xenografts showed vital parathyroid tissue inside intact microcapsules surrounded by a small rim of fibroblasts. Avital fibrotic remnants were demonstrated in animals with non-encapsulated parathyroid tissue. Thus, we have established the feasibility of microencapsulation of human parathyroid tissue, preserving its viability over long periods in vivo even if xenotransplanted. In combination with an improved tissue culture method, transplantation of human parathyroid tissue and maintenance of its physiological function is reproducibly achieved without postoperative systemic immunosuppression over the highest transplantation barrier. This may be a crucial step towards the first clinical application of this method.

Biocompatible Encapsulation Materials: Fundamentals and Application

Despite many advances in transplantation technology, immune rejection of allograft and xenograft tissue remains a problem. Patients still face a lifetime of immunosuppression therapy to inhibit this natural response. An alternative approach is immunoisolated transplantation in which exogenous tissue is encapsulated into semipermeable, artificial membranes. Thus, the tissue is physically isolated from contact with immune effector cells and humoral factors (Lanza et al 1992a Lanza and Chick 1997, Colton 1995). This new technology is highly advantageous, because in principle it does not induce an immune response, thus avoiding immunosuppressant therapy (Colton 1995, Lanza et al 1992a, Lanza and Chick 1997, Lim and Sun 1980). The development of biocompatible materials that can be used in encapsulation of tissue for therapeutic purposes is a rapidly growing field of modern biotechnology. However, most basic materials used for cell encapsulation were designed and developed for various industrial purposes and are usually not commercially available at the high purity and chemical standardization required for medical use (Greco 1994, Hubbell 1995). Impurities may contribute to device failures as well as to undesairable host responses to many implants.

TRANSPLANTATION OF PARATHYROID TISSUE IN EXPERIMENTAL HYPOPARATHYROIDISM :IN VITRO AND IN VIVO FUNCTION OF PARATHYROID TISSUE MICROENCAPSULATED WITH A NOVEL AMITOGENIC ALGINATE

Microencapsulation of tissues is an alternative to postoperative immunosuppression in transplantation. In 1994 iso-, allo- and xenotransplantation of microencapsulated parathyroid tissue was achieved in vivo. However, continued analysis of the coating substance (an alginate) determined mitogenic properties. Here, we report on the in vitro and in vivo function of parathyroid tissue microencapsulated with a novel amitogenic alginate suitable for use in humans. To assess in vitro function, parathyroid tissue encapsulated with mitogenic and amitogenic alginate was exposed to rising concentrations of calcium. For in vivo experiments, it was isotransplanted into parathyroidectomized rats. PTH release into medium and PTH serum levels as well as calcium levels of recipient rats were analyzed and compared to native (non-microencapsulated) tissue and empty capsules, respectively. In vivo, transplants were excised and subjected to histologic examination six months after trans-plantation. In vitro, parathyroid tissue encapsulated with amitogenic alginate releases approximately half of the PTH of native tissue, not different from tissue encapsulated with the mitogenic alginate. In vivo, the novel alginate preserved parathyroid function similar to that of native tissue over the six month period resulting in complete reversal of hypoparathyroidism. Correspondingly, histologic examination revealed vital parathyroid tissue in intact microcapsules. By establishing in vitro function and successful long-term transplantation, we have documented the principle of microencapsulation of parathyroid tissue to be effective also with the novel amitogenic alginate, which is suitable for clinical use.

Octreotide: effective treatment for hyperparathyroidism? A prospective, randomized, controlled clinical trial.

BACKGROUND Recent studies suggest a role for somatostatin in the medical treatment of hyperparathyroidism. In a prospective, randomized, controlled, triple blinded clinical trial in patients with primary or secondary hyperparathyroidism, we evaluated the response of biochemical parameters relevant in hyperparathyroidism to the somatostatin analog octreotide. METHODS Forty patients each with primary or secondary hyperparathyroidism were studied. Among other parameters, serum calcium and serum phosphate, parathyroid hormone, calcitonin, osteocalcin, and octreotide were assessed before and repeatedly for 4 hours after a single intravenous application of 200 micrograms octreotide or placebo. Subsequent to operation, which was performed on all patients, somatostatin-receptor autoradiography of parathyroid tissue was performed. RESULTS After administration of octreotide, which resulted in an increase of plasma levels to pharmacologic levels, no significant changes in any of the biochemical parameters investigated for were observed. Multivariate analysis did not identify patient subpopulations that responded to either drug or placebo (p > 0.05). Forty-five percent of patients receiving octreotide reported side effects: Parathyroid tissue samples of patients with primary or secondary hyperparathyroidism were negative for somatostatin-receptor expression. CONCLUSIONS Octreotide has no discernible effect on biochemical parameters of patients with primary or secondary hyperparathyroidism. Absence of somatostatin receptors, together with lack of octreotide effects, suggests that somatostatin is not effective in the medical therapy of hyperparathyroidism.

Transplantation of Microencapsulated Parathyroid Tissue: Clinical Background, Methods, and Current Status of Research

While autotransplantation of parathyroid tissue is a well-established clinical procedure (Wells et al 1974), allotransplantation of the parathyroid gland still is at the level of experimental surgery. (See Table 20.1.)

FGFR-ERK signaling is an essential component of tissue separation.

Formation of a constriction and tissue separation between parent and young polyp is a hallmark of the Hydra budding process and controlled by fibroblast growth factor receptor (FGFR) signaling. Appearance of a cluster of cells positive for double phosphorylated ERK (dpERK) at the late separation site indicated that the RAS/MEK/ERK pathway might be a downstream target of the Hydra Kringelchen FGFR. In fact, inhibition of ERK phosphorylation by the MEK inhibitor U0126 reversibly delayed bud detachment and prevented formation of the dpERK-positive cell cluster indicating de novo-phosphorylation of ERK at the late bud base. In functional studies, a dominant-negative Kringelchen FGFR prevented bud detachment as well as appearance of the dpERK-positive cell cluster. Ectopic expression of full length Kringelchen, on the other hand, induced a localized rearrangement of the actin cytoskeleton at sites of constriction, localized ERK-phosphorylation and autotomy of the body column. Our data suggest a model in which (i) the Hydra FGFR targets, via an unknown pathway, the actin cytoskeleton to induce a constriction and (ii) FGFR activates MEK/ERK signaling at the late separation site to allow tissue separation.

Konservative Therapie der anorektalen Inkontinenz

1 Klinik für Viszeral-, Thoraxund Gefäßchirurgie, Philipps-Universität Marburg. Mit einer aktuellen Prävalenz von 7% bei geschätzten 1,5–3,9 Millionen Betroffenen gehört die anale Inkontinenz zu den häufigsten Krankheitsbildern in der Koloproktologie. Neben Kenntnissen über die Komplexität der Ätiopathogenese und Diagnostik gewinnt bei einem immer älter werdenden Patientenkollektiv, mit folglich steigendem perioperativen Risiko, die Ausschöpfung der konservativen Behandlungsoptionen immer mehr an Bedeutung. Dieser Beitrag soll ausschließlich wissenschaftlich Gesichertes vermitteln. Zunächst wird eine systematische Übersicht über die Möglichkeiten konservativer Therapie gegeben (Abbildung 1). Der Fülle z.T. verwirrender Fachtermini (Abbildung 2) sollen klare Definitionen wesentlicher Grundbegriffe entgegengestellt werden (Abbildungen 3, 4 und 6). Wir möchten damit u.a. das Verständnis eröffnen, was die aktuelle Gerätegeneration kann (Abbildung 5) und was noch nicht. So ist inzwischen wissenschaftlich belegt, dass sich sportphysiologische Basiserkenntnisse, wie sollte es anders sein, auch auf die Konditionierung des muskulösen Beckenbodens übertragen lassen. Bisher wird Elektrostimulation statisch betrieben (Abbildung 7). Im Hochleistungssport wird seit Jahren, unter strenger Erfolgskontrolle, mit systematisch wechselnder Intensität trainiert (Abbildung 9). Mit der Umsetzung dieser Grundregeln definiert sich die dynamische Elektrostimulation (Abbildung 10). Sie ist, evidenzbasiert, sicher der unphysiologischen, monotonen, unkontrollierten Therapie überlegen (Abb. 11). Leider ist es der deutschen Industrie, im Gegensatz zur wieder einmal flexibleren internationalen Konkurrenz, derzeit noch nicht gelungen, die gesicherten wissenschaftlichen Daten, die sich in der Literatur diesbezüglich häufen, in eine möglichst einfach zu handhabende Gerätekonfiguration zusammenzufassen. Jeder Therapeut weiß, dass der, zumeist reife, Patient nicht das komplizierte Gerät mit mehreren unverständlichen Knöpfchen, digital und mit vielen unsinnigen Lämpchen will. Er möchte einen Schalter und ein Gerät, das ihn führt – die Simplifizierung ist aber meistens besonders schwierig. Sie wäre für den Patienten hilfreich, denn niedrige Schweregrade der analen Inkontinenz lassen sich so, auch ohne oftmals kostspielige Operation, in den meisten Fällen befriedigend therapieren (Abbildung 12). Voraussetzung dafür ist aber die Ausschöpfung aller gesicherter Elemente konservativer Therapie der analen Inkontinenz, d.h. nicht alleiniges Sphinktertraining (Abbildung 8).