Process engineering design of pathological waste incinerator with an integrated combustion gases treatment unit.

Management of medical wastes generated at different hospitals in Egypt is considered a highly serious problem. The sources and quantities of regulated medical wastes have been thoroughly surveyed and estimated (75t/day from governmental hospitals in Cairo). From the collected data it was concluded that the most appropriate incinerator capacity is 150kg/h. The objective of this work is to develop the process engineering design of an integrated unit, which is technically and economically capable for incinerating medical wastes and treatment of combustion gases. Such unit consists of (i) an incineration unit (INC-1) having an operating temperature of 1100 degrees C at 300% excess air, (ii) combustion-gases cooler (HE-1) generating 35m(3)/h hot water at 75 degrees C, (iii) dust filter (DF-1) capable of reducing particulates to 10-20mg/Nm(3), (iv) gas scrubbers (GS-1,2) for removing acidic gases, (v) a multi-tube fixed bed catalytic converter (CC-1) to maintain the level of dioxins and furans below 0.1ng/Nm(3), and (vi) an induced-draft suction fan system (SF-1) that can handle 6500Nm(3)/h at 250 degrees C. The residence time of combustion gases in the ignition, mixing and combustion chambers was found to be 2s, 0.25s and 0.75s, respectively. This will ensure both thorough homogenization of combustion gases and complete destruction of harmful constituents of the refuse. The adequate engineering design of individual process equipment results in competitive fixed and operating investments. The incineration unit has proved its high operating efficiency through the measurements of different pollutant-levels vented to the open atmosphere, which was found to be in conformity with the maximum allowable limits as specified in the law number 4/1994 issued by the Egyptian Environmental Affairs Agency (EEAA) and the European standards.

Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep.

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.

A kinetic model for the homing and migration of prenatally transplanted marrow.

Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (10(6) LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.

In utero bone marrow transplantation induces donor-specific tolerance by a combination of clonal deletion and clonal anergy.

BACKGROUND/PURPOSE: In utero bone marrow transplantation can induce donor-specific tolerance to postnatal solid organ transplantation, although the mechanisms remain poorly defined. In this study, we investigated the role of clonal deletion and clonal anergy in the maintenance of tolerance in a murine model of in utero bone marrow transplantation. METHODS: DBA/2 mice (MIs(a+)) were used as donors of adult bone marrow, and 14-day-gestation fetal Balb/c mice (MIs(a-)) were used as recipients. Tolerance was defined by donor-specific skin graft survival for more than 8 weeks. Clonal deletion was assessed by flow cytometry for Vbeta6 T cell receptor usage. A tolerant animal demonstrating partial deletion of CD4+/Vbeta6+ T cells and a nontolerant animal were selected for analysis of clonal anergy by a proliferation assay using plate-bound anti-Vbeta6 antibody for stimulation with or without exogenous interleukin-2 (IL2). RESULTS: Vbeta6+ splenocytes constituted 6.32% of CD4+ T cells in the tolerant animal compared with 9.19% in the nontolerant animal, demonstrating incomplete clonal deletion in the tolerant animal. Stimulation with plate-bound anti-Vbeta6 induced a good proliferative response in the nontolerant animal but a significantly attenuated response in the tolerant animal (P< .001), which was abrogated by the addition of IL2. CONCLUSIONS: In this murine model of in utero bone marrow transplantation, the tolerant state is characterized by partial clonal deletion of donor reactive T cells and clonal anergy of nondeleted donor reactive T cells. The anergic state can be abrogated by exogenous IL2, suggesting that the mechanism of anergy is a deficiency of IL2 production.

Persistent postnatal transgene expression in both muscle and liver after fetal injection of recombinant adenovirus.

BACKGROUND/PURPOSE: Immune responses to both vector and transgene antigens have limited the efficacy of postnatal gene therapy. We hypothesize that the fetal period may offer immunologic and developmental advantages for successful gene therapy. In this study we examined the efficacy, persistence, and immunologic effects of recombinant adenovirus after intramuscular delivery into fetal mice. METHODS: E1-deleted adenovirus (AdCMVlacZ) containing the beta-galactosidase marker gene was used for injection. Fetal Balb/c mice (14 to 15 days' gestation) were injected with AdCMVlacZ in 10-microL volume in either the shoulder or hindlimb musculature. Animals were killed at 18 to 20 days' gestation and up to 4 months postnatally for analysis of transgene expression and adenoviral genome persistence. RESULTS: Fetuses were injected with doses of AdCMVlacZ from 1 x 10(8) to 2 x 10(10) viral particles (n = 80). Optimal survival rate was 83% at 18 to 20 days' gestation and 55% at 4 weeks of age using a dose of 1 x 10(9) particles. Expression of beta-galactosidasae at 18 to 20 days localized to multiple muscle groups surrounding the site of injection, as well as bone marrow stroma, liver, lung, and dorsal root ganglia. Persistent muscle and liver transgene expression was observed for as long as 16 and 8 weeks, respectively, after injection. The pattern of liver expression was confined to discrete foci of hepatocytes, which appeared to increase in size in older animals. No histological evidence of muscle or liver inflammation was observed at any time after injection. No neutralizing antibodies were observed postnatally. CONCLUSIONS: Our results confirm that gene therapy in the fetus may be advantageous. Distribution of vector in the fetus at the site of injection is clearly broader than in the adult setting. Furthermore, the absence of immune response and persistence of transgene expression suggests that fetal exposure to foreign transgene and vector antigens may induce tolerance. Although we have not proven genomic integration, the histological appearance of transgene expression in the liver supports this conclusion. By understanding the mechanisms that underlie persistent transgene expression, fetal gene therapy may become a feasible strategy for the treatment of fatal genetic diseases.

Fetal hematopoietic stem cell transplantation.

In utero hematopoietic stem cell transplantation (IUHSCTx) is a promising approach for the treatment of a potentially large number of fetuses affected by congenital hematologic disorders. With technical and molecular advances in prenatal diagnosis, the majority of these diseases can now be diagnosed early in gestation, allowing consideration of prenatal treatment. In addition, technical advances in fetal imaging and intervention make it possible to perform the transplants with relatively minimal risk. It, therefore, stands to reason that there is increasing interest in performing in utero hematopoietic stem cell transplantation at many fetal treatment centers. Although the approach remains experimentally promising, expansion of clinical application will depend on improved understanding of the biological barriers to engraftment in the fetus as well as the development of effective clinical strategies based on the hematopoietic biology of individual disorders. This article presents the current status of this emerging therapeutic approach.

Microchimerism and tolerance after in utero bone marrow transplantation in mice.

BACKGROUND: Donor-specific tolerance has been induced after both fetal and neonatal hematopoietic stem cell (HSC) transplantation in mice. However, the relationship between hematopoietic microchimerism and tolerance in these models has not been defined due to the insensitivity of donor cell detection methodology. To address this problem we developed a semiquantitative polymerase chain reaction (PCR)-based assay for detection of microchimerism after major histocompatibility (MHC) class I disparate HSC transplantation. This assay was used to examine the relationship between microchimerism and tolerance after fetal and neonatal transplantation of fully allogeneic bone marrow cells. MATERIALS AND METHODS: C57BL/6 mice (H2-Kb) were used as adult bone marrow donors and Balb/c mice (H2-Kd) were used as fetal or newborn recipients. A dose of 10(10) BM cells/kg was injected intraperitoneally into recipient animals. Peripheral blood of animals which survived beyond 3 weeks of age was analyzed by PCR for the presence of donor MHC class I DNA. Tolerance was tested by placement of donor-specific skin grafts after determination of chimerism status. RESULTS: Our assay was found to be specific for H2-Kb donor cells in an H2-Kd background with a sensitivity of <0.0001%. Of 49 animals injected in utero 19 (38%) had donor DNA present in peripheral blood at low levels (<0.1%) whereas only 1 of 18 neonatally injected animals had detectable donor cells (P < 0.01). Tolerance to donor-specific skin grafts was found in 6 of 9 animals which were chimeric after in utero HSC transplantation whereas none of the 18 neonatally injected animals including the chimeric animal were tolerant. CONCLUSIONS: Our results indicate the following. (1) Hematopoietic microchimerism can be detected by PCR in peripheral blood after in utero injection of fully allogeneic HSCs. (2) Fetal injections yield a higher incidence of microchimerism than newborn injections. (3) Tolerance can be induced across a fully allogeneic barrier by in utero HSC transplantation and this is associated with the presence of peripheral blood microchimerism.

Reduction mammaplasty: the results of avoiding nipple-areolar amputation in cases of extreme hypertrophy.

In extreme cases of breast hypertrophy, amputation of the nipple-areolar complex and transplantation during reduction mammaplasty has been advocated to avoid nipple necrosis. We report our experience with 172 patients having inferior breast pedicle reduction without amputation of the nipple-areolar complex. Mean total weight of resected tissue was 1,946 g (548 to 5,100 g), with a mean nipple-areolar transposition of 10 cm (0.5 to 23 cm). Dividing patients into four groups by weight of resection, we compared complication rates. In this series, where nipple-areola amputation was avoided, there was a 99.6% survival rate of the nipple-areolar complex with 97.1% retention of nipple sensibility. Patients with extreme breast hypertrophy (3,000 g resected tissue) experienced no increase in complications when compared to smaller reductions. In most cases of gigantomastia, amputation of the nipple can be avoided using the inferior breast pedicle technique. Size of breast resection alone should not determine the fate of the nipple.