Amnion-derived multipotent progenitor cells support allograft tolerance induction.

Donor-specific immunological tolerance using high doses of bone marrow cells (BMCs) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease remains a risk. Here, we demonstrate that the co-infusion of limited numbers of donor unfractionated BMCs with human amnion-derived multipotent progenitor cells (AMPs) 7 days post-allograft transplantation facilitates macrochimerism induction and graft tolerance in a mouse skin transplantation model. AMPs + BMCs co-infusion with minimal conditioning led to stable, mixed, multilineage lymphoid and myeloid macrochimerism, deletion of donor-reactive T cells, expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cells (T(regs)) and long-term allograft survival (>300 days). Based on these findings, we speculate that AMPs maybe a pro-tolerogenic cellular therapeutic that could have clinical efficacy for both solid organ and hematopoietic stem cell transplant applications.

Immune status of recipients following bone marrow-augmented solid organ transplantation.

It has been postulated that the resident "passenger" leukocytes of hematolymphoid origin that migrate from whole organ grafts and subsequently establish systemic chimerism are essential for graft acceptance and the induction of donor-specific nonreactivity. This phenomenon was augmented by infusing 3 x 10(8) unmodified donor bone-marrow cells into 40 patients at the time of organ transplantation. Fifteen of the first 18 analyzable patients had sequential immunological evaluation over postoperative intervals of 5 to 17 months, (which included 7 kidney (two with islets), 7 liver (one with islets), and one heart recipient). The evolution of changes was compared with that in 16 kidney and liver nonmarrow controls followed for 4 to 5 months. The generic immune reactivity of peripheral blood mononuclear cells (PBMC) was determined by their proliferative responses to mitogens (PHA, ConA). Alloreactivity was measured by the recipient mixed lymphocyte reaction (MLR) to donor and HLA-mismatched third-party panel cells. Based on all 3 tests, the recipients were classified as donor-specific hyporeactive, intermediate, and responsive; patients who were globally suppressed made up a fourth category. Eight (53%) of the 15 marrow-treated recipients exhibited progressive modulation of donor-specific reactivity (3 hyporeactive and 5 intermediate) while 7 remained antidonor-responsive. In the nonmarrow controls, 2 (12.5%) of the 16 patients showed donor-specific hyporeactivity, 10 (62.5%) were reactive, and 4 (25%) studied during a CMV infection had global suppression of responsiveness to all stimuli.

TGF-beta 1 pretreatment impairs the allostimulatory function of human bone marrow-derived antigen-presenting cells for both naive and primed T cells.

Transforming growth factor-beta (TGF-beta) exhibits strong antiproliferative effects upon lymphocytes and inhibits many of the effector functions of activated immune cells. However, its influence on the inductive phase of immune responses, and in particular its effect on antigen-presenting cells (APC), has not been well studied. In this investigation, we examined the influence of human TGF-beta 1 on the antigen-presenting function of human bone marrow (BM)-derived APC propagated in liquid culture for 11-17 days in response to granulocyte/macrophage colony-stimulating factor (GM-CSF). These cells were predominantly macrophages, accompanied by a minor population of dendritic cells. TGF-beta 1 had no effect upon the allostimulatory function of vertebral body whole BM cells cultured for 3-5 days in GM-CSF. However, it markedly reduced the allostimulatory capacity of BM-derived APC exposed to the cytokine for the last 3 days of culture. This inhibitory action could not be ascribed to cytokine 'carry-over', or to any consistent changes in the expression of cell surface molecules implicated in antigen presentation (HLA-DR), intercellular adhesion (ICAM-1; CD54), or costimulatory activity (B7-1; CD80). Mechanisms that may underlie the inhibitory action of TGF-beta on APC function and the immunologic and possible clinical implications of the findings are discussed.

Optimization of an automated DNA purification protocol for neonatal screening.

CONTEXT: Collection of blood from newborns is a standard clinical procedure used for genetic screening. Typically, blood from a heel prick is absorbed onto standard collection paper and dried before analysis of metabolites, proteins, hormones, and more recently DNA. OBJECTIVE: To evaluate strategies to purify DNA for use with automated workstations. DESIGN: Two factors were used to evaluate several DNA purification protocols: residual heme contamination and amplification yield. The protocol that produced DNA with the lowest heme content and the highest amplification yield was selected. In combination with those two performance factors, the protocol with the fewest number of steps was chosen to reduce reagent use and processing time. SETTING: Industrial research and development laboratory. RESULTS: Robust amplification of DNA isolated from dried blood spots was demonstrated using both fluorescence and agarose gel-based detection methods. In addition, the samples had consistent DNA volumes and had no detectable cross-contamination. Suggested instrument settings, equipment, and supplies were included for automated processing of DNA from dried blood spots. CONCLUSION: A 4-step DNA processing protocol was developed for dried blood spots. The protocol could be performed in either a manual or automated format, making it possible to process hundreds of samples in 1 day.

DNA microarray technology for neonatal screening.

Modern molecular biology, owing much to the Human Genome Initiative, has elucidated many of the genetic mechanisms underlying heritable metabolic disease. While the use of molecular methods has flourished in research laboratories, complexity and cost have limited their utility in newborn screening. Newborn blood cards provide high quality DNA samples able to provide reliable support to highly multiplexed polymerase chain reactions (PCR). New manufacturing processes have reduced the cost of DNA microarray technology to the point where it is a practical tool for population screening. In a single assay, a DNA microarray facilitates the co-detection of amplification products diagnostic for several genetic diseases. High throughput is achieved with automation at every step, from DNA extraction to detection of hybrids. We suggest that it is both feasible and practical to develop a first-tier newborn screening protocol based upon multiplex PCR and analysis of amplification products using DNA microarrays. Initial data utilizing the model systems of sickle cell disease, alpha-1-antitrypsin deficiency and Factor V Leiden will be reported.

[Prevalence of malignant neoplasms in the Tarnów region in the years 1976-1992]. / Wystepowanie nowotworów zlosliwych w województwie tarnowskim w latach 1976-1992.

In the paper the prevalence and kinds of neoplasms in Tarnów region in the years 1976-1992 was presented. An increase in mortality, particularly in men, probably connected with the influence of carcinogenic environmental factors, especially smoking cigarettes, was observed. The most frequent malignant neoplasm in men was lung cancer whereas in women-breast cancer. An assessment of occurrence of malignant neoplasms in various part of Tarnów region was performed. In comparison with all Polish population as well as a majority of other regions' population, the Tarnów region is characterised by a little lower incidence of malignant neoplasms.

Tumor necrosis factor-alpha production by alveolar macrophages in heart-lung transplant recipients.

Tumor necrosis factor-alpha (TNF alpha) is a cytokine produced by mononuclear cells that amplifies inflammation and modulates expression of Class I and Class II histocompatibility antigens. Because of these properties, this cytokine may exert a central role in both the defense and the rejection of the transplanted lung. Utilizing an ELISA technique, we measured TNF alpha in vivo and in vitro in several compartments of lung transplant recipients and in normal subjects that included serum, bronchoalveolar lavage fluid (BAL), and media conditioned by alveolar macrophages (AM) and by autologous peripheral blood monocytes (PBM). Overall, stimulated production of TNF alpha by AM from lung recipients in vitro was less than that of cells from normal subjects in response to lipopolysaccharide (LPS) challenge, and stimulated production of TNF alpha by AM harvested during conditions of infection or acute and chronic rejection was less than that by cells from healthy lung recipients. AM from normal subjects and allograft recipients produced substantially more TNF alpha than autologous PBM, but release in vitro by PBM from recipients was the same as that from cells of normal subjects who were not immunosuppressed. Thus, systemic immunosuppression does not seem to affect the production of TNF alpha by PBM in vitro, but it may reduce production by AM, indicating different effects of immunosuppression on different compartments of mononuclear cells. This mediator was not detected at elevated levels in serum, and it was undetectable in BAL fluid. We conclude that AM from lung recipients are capable of producing TNF alpha, which would influence the defense and immunogenicity of the allograft.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical significance of cytomegalovirus-specific T helper responses and cytokine production in lung transplant recipients.

Cytomegalovirus (CMV) disease continues to be a major problem for lung transplant recipients. In CMV-seropositive individuals, we detected two types of CMV-specific responses: a self-restricted response stimulated by soluble CMV antigen (sCMV-Ag) and a non-self-restricted response induced by CMV-infected cells (cCMV-Ag). Lung transplant recipients who develop the CMV-specific self-restricted T helper response have a low risk of recurrent CMV disease. In contrast, during CMV disease, lung transplant recipients exhibit only the non-self-restricted T helper responses. We characterized the T cell activation and the kinetics of cytokine production of sorted CD4+ and CD8+ T cells from PBLs of CMV seropositive donors. The two types of CMV antigens induced cytokine production in both T cell subsets. We also performed competitive RT-PCR for Granzyme B (GB) in BAL cells of lung transplant recipients prior to, during and following CMV disease. CMV disease was associated with increase in GB gene expression when was accompanied by acute cellular rejection while it remained low in patients with CMV disease that did not have a complicated course. In summary, CMV-activated T cells within the allograft may produce inflammatory cytokines and effector molecules that may promote allograft rejection.

Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified by MS/MS-based prospective screening of newborns differ from those observed in patients with clinical symptoms: identification and characterization of a new, prevalent mutation that results in mild MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.