Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group.

An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

Meeting the bereavement needs of kids in patient/families--not just playing around.

Children grieve differently than adults. Although the unit of care in hospice is the patient and family, emphasis is often on the grown members of the family and the anticipatory grief and bereavement needs of children and adolescents are sometimes not met adequately. In this paper, hands-on strategies for working with children both before and after a significant death are presented, as well as appropriate context information on the grieving process for youth under age 18.

Lamotrigine for the treatment of epilepsy in childhood.

OBJECTIVE: In this multicenter study, the efficacy and tolerability of lamotrigine were assessed in 285 children less than 13 years of age, recruited from 37 centers in 11 countries. METHODS: Pooled data from five open add-on studies have been analyzed. All the children had treatment-resistant epilepsy and most had two or more seizure types. Seizure frequency and global evaluation were assessed at the end of four successive 12-week periods of therapy. RESULTS: Seizure frequency was reduced by 50% or more in one third of the patients. Lamotrigine was effective in all seizure types examined, particularly for typical and atypical absence seizures. Atonic seizures also responded well. Improvement was well maintained during the treatment period. The maintenance dose had to be adjusted according to concomitant medication; dose ranges were 1 to 5 mg/kg per day for children taking valproate and 5 to 15 mg/kg per day for those not taking valproate. The commonest reported adverse experiences were somnolence, rash, vomiting, and seizure exacerbations. Adverse experiences led to withdrawal of treatment from 36 patients (12.6%). CONCLUSIONS: These results indicate that lamotrigine is well tolerated and is effective for a broad range of seizure types, especially absence seizures and atonic seizures.

Stimulation of in vitro growth of Treponema denticola by extracellular growth factors produced by Porphyromonas gingivalis.

Cell-free culture filtrates of Porphyromonas gingivalis grown in Wilkins-Chalgren broth stimulated the growth of six strains of Treponema denticola in 1186 broth when compared with the effect of uninoculated WC. The pH of the 1186 broth was not altered by the addition of either culture filtrate or WC, and all media were fully reduced prior to inoculation with T. denticola. Growth was also stimulated by factors precipitated from the culture filtrate with 90% (NH4)2SO4, 50% cold ethanol, or 50% cold acetone, and by factors retained after dialysis of the culture filtrate through a membrane with a molecular weight cut-off of 50 kDa. Growth factor activity was eliminated by heating of the culture filtrate at 55 degrees C for 4 h. An ether extract of the culture filtrate containing acetic, butyric, isobutyric, isovaleric, propionic, and phenylacetic acids did not stimulate growth. Since subgingival plaque from periodontal pockets colonized with T. denticola also contains P. gingivalis, certain extracellular proteins with molecular weights greater than 50 kDa produced by P. gingivalis may act as growth factors for T. denticola in the microenvironment of the periodontal pocket.

Suppression of kidney allograft rejection across full MHC barriers by recipient-specific antibodies to class II MHC antigens.

The aim of these studies was to see if recipient-specific antibodies to class II MHC antigens might be effective in suppressing kidney graft rejection in rats. For these experiments, the polymorphic BMAC-4 mouse IgG1 monoclonal antibody to RT1-D class II MHC antigens was raised. This antibody reacts with the DA, LEW, PVG, and SHR strains, but not the BN or WAG strains, and is therefore recipient-specific in the WAG to PVG combination. Initial in vivo titrations demonstrated that 1 ml doses of the BMAC-4 and also of the MRC OX6 (monomorphic mouse IgG1 anti-RT1-B class II) antibody resulted in the maintenance of free antibody levels in blood for greater than 24 hr. Treatment of PVG recipients of WAG kidney allografts with the BMAC-4 antibody, but not the MRC OX6 antibody, resulted in greatly prolonged graft survival. To examine possible mechanisms, several experiments were performed. After intravenous injection, the antibody was found to have ready access to the connective tissues of nonlymphoid organs, to the red and white pulp of the spleen, and to the medulla of lymph nodes. However, there was poor early access to the cortex and paracortex of lymph nodes. Both MRC OX6 and BMAC-4 could completely suppress PVG anti-WAG and WAG anti-PVG mixed lymphocyte culture reactions. Both antibodies were also equally effective for opsonisation of class II-positive cells from the blood circulation. However, only the recipient-specific, anti-RT1-D BMAC-4 antibody suppressed graft rejection. Thus, while the BMAC-4 antibody is likely to have had a variety of different effects on RT1-D positive recipient cells, the locus specificity of the immunosuppression is consistent with an important component of those effects being the blocking of presentation of WAG donor alloantigens by PVG RT1-D class II antigens on PVG antigen-presenting cells.

Electroporation technique of DNA transfection.

Electroporation is a simple and rapid procedure by which DNA may be transferred into cells. Essentially, a high voltage pulse is applied to a suspension of cells and DNA placed between electrodes in a suitable cuvet. It is thought that this pulse induces local areas of cell-membrane breakdown, or pores, through which the DNA then enters the cell. Once these pores have resealed, normal cell functions can continue. Since this transfection method involves a physical effect of the delivered pulse on the cell membrane, it can potentially be used for most cells, independent of their phagocytic capacity as required for transfection by the calcium phosphate coprecipitation technique (see Chapter 2 , this vol.).

Characterization of the tissue macrophage and the interstitial dendritic cell as distinct leukocytes normally resident in the connective tissue of rat heart.

Immunohistological studies with a mouse anti-rat macrophage mAb (BMAC-5) demonstrated the presence of numerous positive cells in the interstitial connective tissues of many organs. The pattern resembled that seen with anti-MHC class II antibodies, with the striking exception that BMAC-5+ cells were rare or absent in the portal triad, the islets of Langerhans, and the kidney. Double-labeling fluorescence studies were therefore performed in rat heart using the BMAC-5 mAb in combination with rabbit antisera to pure rat class II MHC antigens and pure rat leukocyte common (CD45) antigens. The tissue macrophages in heart were identified as BMAC-5+, MHC class II-negative, leukocyte common antigen-positive cells. They could be distinguished from the BMAC-5-, MHC class II-positive, leukocyte common antigen-positive interstitial dendritic cells. Moreover, 7 d after lethal irradiation, the class II-positive interstitial dendritic cells had completely disappeared from heart, whereas the BMAC-5+ macrophages were present in undiminished numbers. These studies strongly suggest that the interstitial dendritic cell and the tissue macrophage represent two distinct populations of leukocytes within the connective tissues of antigenically secluded organs such as the heart. They have potentially important implications for the physiology of the immune system, as well as for autoimmunity and transplantation.

Donor class I and class II major histocompatibility complex antigen expression following liver allografting in rejecting and nonrejecting rat strain combinations.

Orthotopic liver allografts in the nonrejecting DA-to-PVG strain combination and in the DA-to-LEW strain combination were studied at various times after transplantation for donor class I and class II MHC expression using immunohistological techniques and quantitative analyses. DA-to-DA isografts were also studied. In the isografts, weak class I induction on hepatocytes and biliary epithelium was noted from day 5, and this persisted to day 15, the last time point examined. In DA-to-PVG allografts, class I induction also appeared on hepatocytes and biliary epithelium from day 5, but was more intense than in the isografts. Nevertheless, the induction was patchy within most grafts, and in some grafts was not prominent. Quantitative absorption analyses demonstrated that the maximum increase in donor class I expression was only 3-fold over the normal liver. In the strong DA-to-LEW combination, class I induction on hepatocytes seemed to appear earlier, beginning at day 3, and was more uniform and intense than in the DA-to-PVG model from day 5. In the isografts, there was no induction of class II antigens on hepatocytes or biliary epithelium at any stage, but from days 5 to 15 there was a marked increase in the number of isolated, class II-positive cells in the hepatic lobule, probably representing class II induction in the Kupffer cells of the isografts. In DA-to-PVG allografts, biliary epithelium became class II-positive from day 5, and this persisted to day 30, the last time point examined. Weak but definite class II induction was seen on some hepatocytes from day 5 through day 30. However, the majority of hepatocytes remained class II-negative. By day 30, there was virtually no donor class II staining the sinusoids, but isolated class II-positive cells of recipient type were seen, the pattern suggesting a replacement of the graft Kupffer cells by recipient Kupffer cells at this stage. By quantitative absorption analysis, donor class II expression in the grafts increased approximately 5-fold. In DA-to-LEW allografts, class II induction was not noticeably different from that seen in the DA-to-PVG model, except that induction of class II antigens on the Kupffer cells possibly appeared earlier in this strain combination.

Bulk purification of a naturally occurring soluble form of RT1-A class I major histocompatibility complex antigens from DA rat liver, and studies of specific immunosuppression.

We describe the bulk purification of a water-soluble form of RT1-A class I MHC antigens from aqueous extracts of DA liver. Using a combination of monoclonal antibody affinity, lentil lectin affinity, and gel permeation chromatography, we were able to obtain large quantities of pure water-soluble RT1-A antigens. Typically, from 40 DA livers, 0.5 mg of pure antigen with antigen activity equivalent to 7 X 10(9) nucleated DA spleen cells was obtained. The water-soluble RT1-A molecule had a discrete heavy chain of 40 kD, linked noncovalently to beta 2 microglobulin. The heavy chain of the water-soluble RT1-A molecule was 5 kD smaller than the membrane-bound form of RT1-A from DA liver membranes. The smaller molecule probably represents a secreted form of RT1-A class I molecules that lack the transmembrane domain (exon 5). Large quantities of this water-soluble RT1-A class I antigen from the DA strain, given intravenously to PVG recipients of DA cardiac allografts by a variety of protocols, did not have any effect on graft survival. The fairly ready availability of milligram quantities of pure class I transplantation antigens should be of considerable value for studies in transplantation.

Identification in rat liver and serum of water-soluble class I MHC molecules possibly homologous to the murine Q10 gene product.

We have identified large quantities of a water-soluble, non-RT1.A class I MHC molecule in the serum of the DA rat strain, with a similar molecule being found in aqueous extracts of DA liver. The non-RT1.A class I molecules have heavy chains of 41 kD, which is smaller than RT1.A class I molecules isolated from liver membranes (45 kD) but larger than water-soluble RT1.A class I molecules previously identified in serum and aqueous extracts of liver and kidney (40 kD). NH3-terminal amino acid sequencing of bulk-purified RT1.A class I molecules and of this novel non-RT1.A class I molecule revealed two substitutions, in the first 25 amino acids, Tyr----His at position 9, and Ala----Ser at position 24. The non-RT1.A class I molecule did not react with any of the well-characterized polymorphic and monomorphic antibodies directed against RT1.Aa class I molecules, but did react with the MRC OX18 antibody. A similar class I molecule could not be identified on liver membranes. The non-RT1.A class I molecule was found in large quantities (approximately 20 micrograms/ml) in the serum of the DA rat strain, and similarly large quantities appeared to be present in the sera of BN, PVG, and LEW.RT1a rats. WAG and LEW.RT1u rats had readily detectable but lower amounts of this molecule in their serum, while LEW and SHR rats had little if any present. This molecule probably represents the rat homologue of the murine Q10 gene product, and is the major class I product in the serum of the DA rat strain.

Prolonged survival of actively enhanced rat renal allografts despite accelerated cellular infiltration and rapid induction of both class I and class II MHC antigens.

Administration of 1 ml of donor whole blood 7 d before renal transplantation produces long-term (greater than 100 d) graft survival in the DA (RT1a) into PVG (RT1c) rat strain combination. Using this model, the pattern and phenotype of infiltrating leukocytes were examined in rejecting and enhanced renal allografts, at days 1, 3, 5, and 7 after transplantation, by immunohistologic techniques. Paradoxically, enhanced grafts showed a more rapid and substantial leukocyte infiltrate, the phenotype of which was similar to that in rejecting grafts except for a reduced number of MRC OX-8+ cells and MRC OX-39+ cells. Graft infiltrating cells and splenocytes from transfused animals showed similar, although modest, levels of both nonspecific cytotoxicity and alloantigen-specific cytotoxicity. Immunohistologic analysis of MHC antigen distribution within the allograft revealed, unexpectedly, that enhanced grafts underwent an accelerated and extensive induction of both donor class I and class II MHC antigens. These findings were confirmed by allospecific quantitative absorption analysis, which showed severalfold increases in class I and class II MHC antigens by day 3 in enhanced grafts but not until day 5 in rejecting grafts. An additional observation was the more rapid disappearance of donor interstitial cells from enhanced grafts. These findings emphasize the overwhelming suppressive effect induced by an organ allograft after preoperative blood transfusion despite the associated induction of large numbers of potential effector cells and increased target antigen density within the graft.

Water-soluble form of RT1.A class I MHC molecules in the kidney and liver of the rat.

The RT1.A (H-2K,D type) class I major histocompatibility complex (MHC) antigens of the rat are well recognized as membrane-bound glycoproteins. In this report, we demonstrate that liver and kidney in the DA rat strain contain large amounts of a water-soluble RT1.A class I molecule with a discrete heavy chain approximately 5 kd smaller than the membrane-bound form. An identical molecule could be identified in DA rat serum. This small class I molecule carries all of the polymorphic antigenic determinants of the RT1.A av1 class I molecule. The water-soluble molecule is readily denatured in its pure form when frozen and thawed, but this does not occur when it is mixed with serum, presumably because of a stabilizing interaction with one or more carrier proteins. The half-life of the class I molecule in serum was measured to be approximately 1.5 h. The LEW rat strain produced detectable but substantially smaller amounts of water-soluble RT1.A molecules. Our studies indicate that RT1.A class I MHC antigens are synthesized and presumably secreted in a smaller water-soluble form by liver, kidney, and possibly other tissues under physiological conditions, a point of considerable interest in view of the immunoregulatory functions of the membrane-bound forms of these molecules.

The effects of cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in the rat.

We have previously reported 5-30-fold increases in the expression of class I and class II major histocompatibility complex (MHC) antigens in rejecting heart and kidney allografts in the DA-to-PVG rat strain combination. We examine here the effects of immunosuppression with cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in this strain combination. Immunohistological studies and quantitative absorption analyses using monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens were used throughout. Heart allografts in cyclosporine-treated rats were examined on day 3,5,7,9,11, and 14 after transplantation, and kidney allografts in cyclosporine-treated rats were examined at day 7. In addition, untreated heart and kidney isografts were studied at days 1,3,5, and 7 after grafting. Immunohistological studies on frozen sections showed that cyclosporine-treated heart and kidney allografts showed no induction of class II MHC antigens, in contrast to untreated heart and kidney allografts. Class I MHC antigen induction did occur in spite of cyclosporine-therapy, but at levels lower than those seen in untreated allografts. Moreover, the pattern and degree of class I induction in the cyclosporine-treated allografts resembled very closely those seen in isografts, and so this induction was, in all probability, a consequence of the transplantation procedure rather than of specific immune responses. We also noted, in the cyclosporine-treated heart allografts, that all donor interstitial dendritic cells had disappeared and been replaced by recipient interstitial dendritic cells by the end of the second week after grafting. In addition, there was no reduction in the class II antigen content of kidney allografts treated for 7 days with cyclosporine. The absence of class II antigen induction in allografts where rejection is effectively suppressed with cyclosporine might be of clinical value in the differential diagnosis between rejection and cyclosporine toxicity in renal transplantation, and between active and inactive cellular infiltrates in heart transplantation.

Detailed analysis and demonstration of differences in the kinetics of induction of class I and class II major histocompatibility complex antigens in rejecting cardiac and kidney allografts in the rat.

In this paper, we analyze in detail donor class I and class II major histocompatibility complex (MHC) antigen induction in heart and kidney allografts in the DA-to-PVG rat strain combination. The immunohistological techniques and quantitative absorption analyses utilize monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens, to enable precise interpretation of the results in terms of the MHC antigens of the graft. Quantitative absorption analyses were performed on homogenates comprising 4-6 allografts pooled at each interval examined (days 1-5 for kidneys, days 3-7 for hearts). In the heart allografts, donor class I antigen induction begins at day 3 after transplantation and proceeds rapidly on the 4th and 5th postoperative days. The maximum level (a 10-fold increase in comparison with normal heart) occurs at day 6, and thereafter the level declines. Donor class II antigen induction in the heart allografts follows a similar pattern. In kidney allografts, it was of particular interest that donor class I induction occurred much more rapidly, being already evident on the first postoperative day, and reaching levels 20-fold greater than normal kidney by day 3. Maximum levels (approximately 30-fold that of normal kidney) of donor class I antigens were reached on days 4 and 5. Donor class II induction, by contrast, developed in kidney grafts with kinetics similar to that seen for class II induction in heart grafts (beginning at day 3 and reaching a maximum of 7-fold over normal kidney at day 5). Immunohistological studies were performed at days 1, 3, 5, and 7 after transplantation. These confirmed the early induction of donor class I antigen in the kidney allografts. In kidney, by the fifth postoperative day, all tubules in the cortex and medulla, and the arteriolar vascular endothelium, were strongly positive for class II antigens. However, the glomerulus, including the glomerular capillary endothelium, remained donor-class-II-negative, except for induction of class II antigens on Bowman's capsule. The endothelium of interstitial capillaries also probably remained class-II-negative. These results have potentially important implications for understanding the development of the rejection response.

Evoked potentials and contingent negative variation during treatment of multiple sclerosis with spinal cord stimulation.

Cervical somatosensory evoked potentials, brainstem evoked potentials, visual evoked potentials, and the cerebral contingent negative variation were recorded in patients with definite multiple sclerosis before, during, and after spinal cord stimulation. Improvements were seen in the cervical somatosensory and brainstem evoked potentials but neither the visual evoked potential nor the contingent negative variation changed in association with spinal cord stimulation. The results indicate that spinal cord stimulation acts at spinal and brainstem levels and that the clinical improvements seen in patients are caused by an action at these levels rather than by any cerebral arousal or motivational effect. The evoked potentials were not useful in predicting which patients were likely to respond to stimulation.