Treatment of Kaposi's sarcoma after solid organ transplantation.

PURPOSE: This retrospective review of all patients who developed Kaposi's sarcoma (KS) after solid organ transplantation at a single institution was undertaken to define the clinical presentation of this malignancy in the setting of iatrogenic immunodeficiency, and to determine the most appropriate treatment for patients in this clinical setting. MATERIALS AND METHODS: The records of 2,099 patients who underwent heart, lung, liver, or kidney transplantation at The Toronto Hospital between January 1, 1981 and June 30, 1995, were reviewed. Twelve patients were identified who developed biopsy-proven KS in the posttransplantation period. Five patients who had disseminated KS who had not responded to either reduction or withdrawal of immunosuppression or to local radiotherapy were treated with combination chemotherapy consisting of doxorubicin 20 to 30 mg/m2, bleomycin 10 mg/m2, and vincristine 2 mg (ABV) administered intravenously every 3 weeks. RESULTS: Eight of 12 patients were male and nine were of Italian origin. KS was limited to a localized area of the skin for only six patients, all after kidney transplantation. Visceral KS was present in three patients. Four of five patients responded to ABV chemotherapy (two complete and two partial remissions). The fifth patient responded to second-line etoposide and cisplatin. The median duration of response was in excess of 13 months (range, 8+ to 45+ months). Toxicity was limited to grade 1 neurotoxicity and grade 1 skin toxicity. CONCLUSION: KS is an uncommon but recognized complication of solid organ transplantation. Combination chemotherapy is a safe and effective treatment for patients with disseminated or visceral KS that fails to respond to changes in immunosuppression.

Phenotypic analysis of migrant, efferent lymphocytes after implantation of cold preserved, peripheral nerve allografts.

Cold-preservation of peripheral nerve allografts in vitro (3 weeks, 5 degrees C) was performed to determine its effect on local lymphocyte migration patterns in vivo. Lymphocyte migration was assessed by continuously monitoring the cell output in the regional lymph for nearly 1 month. Cold-preservation delayed or prevented the typical biphasic increase in efferent lymphocyte output observed after fresh allograft implantation. It also decreased the output of activated lymphocytes (CD 5 and MHC class II positive) compared with that seen in the fresh allograft response. These changes suggest that the host immune response to preserved nerve allografts is altered over a prolonged period in vivo (3 weeks). Cold-preservation may be a useful method of reducing allograft immunogenicity, thereby limiting systemic immunosuppression requirements for the successful clinical utilization of peripheral nerve allografts.

Effect of cold preservation on lymphocyte migration into peripheral nerve allografts in sheep.

Lymphocyte migration into fresh and preserved peripheral nerve allografts was quantitated to assess the effect of cold preservation and freeze-thawing pretreatment on the local immunological response to nerve allografts. Out-bred ewes received multiple 1.5-cm subcutaneous heterotopic peroneal nerve autografts, fresh allografts, and pretreated allografts, implanted within the same recipient. Lymphocyte migration was studied at 7 days by injecting autologous 111indium-labeled lymphocytes intravenously. After 3 hr of recirculation, lymphocyte migration into graft tissue was quantitated by a gamma counter (epm/g, mean +/- SEM). Lymphocyte traffic into fresh nerve allografts (21,623 +/- 3783) increased an average 9.4-fold over the autograft value (2918 +/- 377, P < 0.04). Histologic studies illustrated a marked lymphocytic infiltrate of CD4+ and CD8+ cells and enhanced class I and II MHC expression in fresh allografts, but not in autografts. Short-term cold preservation, for 6 and 12 hr (5 degrees C), enhanced lymphocyte entry into pretreated allograft tissue. Conversely, cold preservation for longer periods (1 and 3 weeks) dramatically reduced lymphocyte migration to values below corresponding autograft levels (783 +/- 100 and 1,252 +/- 120, respectively, P < 0.01). A comparable reduction in lymphocyte migration into nerve allografts was observed after freeze-thawing pretreatment (P < 0.01). Cold preservation of donor allogeneic lymphocytes inhibited their capacity to induce intradermal host lymphocyte migration, implicating passenger lymphocytes as a potential cold-sensitive allogeneic component of the nerve allograft. Assessment of the local response to ovine peripheral nerve allografts, utilizing radiolabeled autologous lymphocytes, demonstrated that cold preservation and freeze-thawing pretreatment significantly reduced lymphocyte migration into nerve allografts. The mechanism(s) of reduced lymphocyte migration may involve inactivation or death of antigen-presenting cells, including passenger lymphocytes.

Acute intestinal graft-versus-host disease in a syngeneic bone marrow transplant recipient.

BACKGROUND: We report a case of intestinal graft-versus-host disease (GVHD) in a syngeneic bone marrow transplant patient. METHODS: Several days after receiving a bone marrow transplant from his identical twin for treatment of non-Hodgkin's lymphoma, a 47-year-old man developed a skin rash and diarrhea. RESULTS: A colonic biopsy on day +15 revealed characteristic changes of acute intestinal GVHD. Molecular studies (microsatellite DNA and HLA sequence-specific primer polymerase chain reaction analyses) confirmed the genotypic identity of donor and host and the improbability of transfusion-associated GVHD. CONCLUSION: This case illustrates that pathological evidence of GVHD does not absolutely require the presence of genetic differences between host and donor and questions existing concepts about the nature of cyclosporine-induced GVHD.

HLA-C locus alleles in patients with psoriatic arthritis (PsA).

The aim of this investigation was to compare the frequency of alleles at the HLA-C locus in patients with psoriatic arthritis to that in the disease free population and to relate the HLA genes to disease phenotype in psoriatic arthritis (PsA). Ninety-four consecutive patients seen in the (PsA) clinic between April and July 1996 and 100 disease-free controls had HLA typing performed by both serologic and molecular techniques. Patients' disease was assessed according to a standard protocol. Fisher's exact test was used to compare the frequency of HLA alleles in the patients and controls. HLA-C determined by PCR-SSP decreased the frequency of non-identified, that is, "blank" (or null) alleles to 6% in patients and controls. HLA-Cw*0602 was present in 32 patients and 18 controls: allele frequency of 17% in the patients versus 9% in the controls (p < 0.01). Amongst patients with psoriatic arthritis, those who carried the HLA-Cw*0602 allele had a significantly earlier mean age of onset of their psoriasis (p = 0.003). This study confirms that molecular DNA techniques improve detection of C-locus alleles. The HLA-Cw*0602 is increased amongst patients with psoriatic arthritis compared to controls. The HLA-Cw*0602 is associated with an earlier age of onset of psoriasis.

HLA-DRB1*04 alleles in psoriatic arthritis: comparison with rheumatoid arthritis and healthy controls.

Psoriatic arthritis (PsA) is an inflammatory arthritis associated with psoriasis usually seronegative for rheumatoid factor. An increased frequency of HLA-DR4 has been noted in PsA, particularly among patients with a rheumatoid arthritis like (RA) arthritis. The aim of the current investigation was to compare HLA-DRB1*04 alleles in patients with PsA, patients with RA, and healthy controls. Sample size calculations based on the frequency of HLA-DR4 suggested that 90 individuals in each patient group would be sufficient to address our question. Therefore, 90 HLA-DRB1*04 positive patients from each patient group underwent high resolution molecular typing and were included in this study. Although HLA-DRB1*0401 was the most frequent allele in all groups, its frequency among the PsA patients was lower than that of RA patients and controls. HLA-DRB1*0402 was higher among patients with PsA. Patients with RA were more likely to have more than one shared epitope allele than either PsA or the healthy control group. HLA-DQB1 alleles did not contribute further information. We suggest that the differences in the class II HLA epitope(s) may also be related to interaction specificity with another molecule functioning in the immune response to a putative arthritogenic antigen and result in differences in disease expression.

A special report: histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors. Quality Assurance and Donor Registries Working Groups of the World Marrow Donor Association.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. Registry and cord blood bank guidelines suggest that, at a minimum, initial HLA typing should be performed for three HLA loci, HLA-A, -B, and -DR, at low resolution/split antigen level. DNA-based testing methods should be utilized for HLA-DR typing. DNA-based testing for HLA-A and -B should replace serologic testing of new volunteer donors and cord blood units as robust protocols and reagents become available to the laboratories. Transplant center guidelines for typing of patient, family and to confirm the HLA types of potential unrelated donors should include, at the minimum, typing HLA-A, B, and -DR loci using primarily DNA-based testing methods at allele level resolution for DRB1 and low resolution/split antigen level for HLA-A and -B. It is strongly recommended that the typing of a patient and the selected donor be performed using the same set of reagents, methodology, and interpretation criteria with fresh tissue samples to ensure HLA identity. Guidelines for laboratory accreditation, approaches to quality assurance and quality control for HLA testing, and suggestions for the format of the HLA database of donor types are also outlined.

Naturally occurring IgG anti-HLA alloantibody does not correlate with HIV type 1 resistance in Nairobi prostitutes.

In an effort to identify an immunological basis for natural resistance to HIV-1 infection, we have examined serum antibody responses to HLA class I antigens in female prostitutes of the Nairobi Sex Workers Study. Anti-HLA antibodies are known to block HIV infectivity in vitro and can be protective against SIV challenge in macaques immunized with purified class I HLA. Thus, it was postulated that broadly cross-reactive alloantibodies recognizing common HLA alleles in the client population might contribute to the prevention of heterosexual transmission of HIV. In fact, 12% of the women were found to have serum IgG antibodies against class I alloantigens. However, this alloantibody did not correlate with the HIV status of the women and was found in a similar proportion of HIV-positive and HIV-resistant women. The observed levels of alloantibody did not increase with HIV infection in susceptible individuals, suggesting that potential antigenic mimicry between HIV and host HLA class I antigens does not significantly increase levels of anti-class I antibodies. The lack of correlation between serum anti-allo-class I HLA antibodies and the risk of sexual transmission indicates that this humoral immune response is unlikely to be the natural mechanism behind the HIV-resistance phenotype of persistently HIV-seronegative women. This result, however, does not preclude the further investigation of alloimmunization as an artificial HIV immunization strategy.

Redistribution of F-actin and large dense-cored vesicles in the human neuroblastoma SH-SY5Y in response to secretagogues and protein kinase Calpha activation.

Our previous studies have shown that noradrenaline release is enhanced by activation of protein kinase Calpha in SH-SY5Y cells. In the present study, we report that activation of protein kinase Calpha leads to (a) partial redistribution of the F-actin cytoskeleton and (b) a 2.5-fold increase in the number of large dense-cored vesicles within 100 nm of the plasma membrane. This redistribution can be prevented by down-regulation of protein kinase Calpha by up to 48 h exposure to phorbol dibutyrate. Treatment with the secretagogues 100 mM KCl, the Ca2+ ionophore A23187 (20 microM) and 1 mM carbachol also leads to a partial disassembly of the F-actin cytoskeleton. This is accompanied by an increase in the number of large dense cored vesicles at the plasma membrane following exposure to KCl and A23187 but not following exposure to carbachol. These results are discussed in relation to the hypothesis that a key step in the enhancement of noradrenaline release following activation of protein kinase Calpha and elevation of intracellular calcium is the movement of large dense cored vesicles to the plasma membrane following partial disassembly of the F-actin cytoskeleton.

Histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors: report from The World Marrow Donor Association. Quality Assurance and Donor Registries Working Groups of the World Marrow Donor Association.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. The ability to identify unrelated stem cell donors in one country for patients in another country requires cooperation and standardization in many areas. The adoption of guidelines for histocompatibility testing, such as those summarized in this report, will facilitate these opportunities and rapidly provide accurately typed donors for patients in need.

Hematopoietic stem cell donor registry strategies for assigning search determinants and matching relationships.

Registries and cord blood banks around the world collect and store the HLA types of volunteers in order to identify matched unrelated donors for patients requiring hematopoietic stem cell transplantation. This task is complicated by the many formats in which HLA types are provided by the testing laboratories (types obtained by serology vs by DNA-based methods; high vs intermediate vs low resolution) and by the need to identify which of these diverse types are most likely to match the HLA assignments of a searching patient as closely as possible. Conversion of the assignments to 'search determinants' may be included within the algorithm used to select and prioritize a list of potentially suitable donors, either as an aid to matching or as a tool to optimize the performance of comparisons within large data files. The strategies used by registries to create search determinants are described. A set of search determinants, utilized by the National Marrow Donor Program, is provided as an example and is intended to initiate further discussion aimed at understanding the process used by each registry with the possibility of developing a standard process among registries worldwide.

Comparison of regeneration across nerve allografts with temporary or continuous cyclosporin A immunosuppression.

The efficacy of short-term immunosuppression in a nerve allograft model was examined by comparing regeneration across peripheral nerve allografts with either temporary (12 weeks) or continuous (30 weeks) cyclosporin A treatment. One-hundred fifty Lewis rats received 2-cm nerve grafts from allogeneic ACI or syngeneic Lewis rat donors and were allocated to the following groups: allogeneic grafts and continuous cyclosporin A, with 18 weeks (20 rats) or 30 weeks (20 rats) of survival after graft placement; allogeneic grafts and temporary cyclosporin A, with 12 weeks (10 rats), 18 weeks (20 rats), or 30 weeks (20 rats) of survival; and control rats with allogeneic and syngeneic grafts, no cyclosporin A, with 12, 18, or 30 weeks (10 rats each) of survival. Functional regeneration across the nerve grafts was serially assessed with walking-track analysis. Endpoint evaluations included electrophysiological, histological, and morphometric studies. Both walking-track and electrophysiological function reached a plateau at a significantly worse level in nonimmunosuppressed allograft recipients than in syngeneic or treated allograft recipients. The group with temporary therapy experienced significant worsening in both motor and electrophysiological function at Week 18, 6 weeks after cyclosporin A withdrawal, compared to the group with continuous treatment. At Week 30, motor and electrophysiological function in the temporary-treatment group recovered to levels similar to those of the syngeneic and continuous cyclosporin A groups. Histological assessment of the graft segments from the temporary cyclosporin A group at 18 weeks showed evidence of rejection, with mononuclear cell infiltration and demyelination; morphometric evaluation demonstrated significantly decreased numbers of nerve fibers in the distal host segment. These histological and morphometric changes were no longer present in the nerves from the temporarily immunosuppressed rats at Week 30. Withdrawal of immunosuppression after successful regeneration through nerve allografts results in short-term graft rejection. Eventual restoration of graft histological and function parameters is comparable to continuously immunosuppressed rats. Temporary immunosuppression of nerve allograft recipients is feasible.

Effects of incremental doses of alfentanil and propofol on the breathing of anaesthetized patients.

Incremental doses of alfentanil and propofol were given to anaesthetized healthy patients undergoing routine orthopaedic surgery. Ventilation was recorded by respiratory inductance plethysmography and analysed by microcomputer. Alfentanil affected primarily expiratory time. The onset of effect lasted 82 s (range 25-173 s); offset was exponential, with a half-life of 146 s (range 62-260 s). Alfentanil also reduced tidal volume, but the effect was less obvious and less consistent. Propofol affected primarily tidal volume. The onset of effect lasted 34 s (range 17-69 s); offset was linear, with a time to 50% recovery of 92 s (range 47-161 s). Propofol had little effect on expiratory time. The drugs had little effect on inspiratory time. Three patients showed periods when the distribution of expiratory times was bimodal; the mechanism for this is unknown.

Hypoxia enhances [3H]noradrenaline release evoked by nicotinic receptor activation from the human neuroblastoma SH-SY5Y.

We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K+]o (100 mM) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [3H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K+-evoked release was unaffected by hypoxia (PO2 approximately 30-38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca2+o with 1 mM EGTA abolished NA release. K+-evoked release was also dramatically reduced in the presence of 200 microM Cd2+ to block voltage-gated Ca2+ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd2+-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca2+ influx through the nAChR pore, or by activating an unidentified Cd2+-resistant Ca2+-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.

Response to interferon therapy: influence of human leucocyte antigen alleles in patients with chronic hepatitis C.

The response to interferon (IFN) therapy in patients with chronic hepatitis C is characterized by normalization of the serum alanine aminotransferase (ALT) activity during treatment, but relapse within 6 months of cessation of therapy is common. Viral characteristics, such as the genotype and viral load, may influence the patient response to IFN. The aim of this study was to examine host factors, namely the genetically determined human leucocyte antigen (HLA) class II alleles, in patients with chronic hepatitis C, and their relationship to the response to IFN therapy. Seventy white patients with chronic hepatitis C, treated with IFN-alpha for 6 months, were enrolled in the study. Serum ALT was measured at the end of treatment to assess short-term response and again 6 months post-treatment to assess sustained response. Sequence-specific primers were used in the polymerase chain reaction (PCR) to amplify genomic DNA isolated from peripheral mononuclear cells. HLA class II alleles were determined by analysis of the amplicon by gel electrophoresis and hybridization of sequence-specific oligonucleotide probes. At the end of treatment, 25 of the 70 patients (36%) had a normal ALT. By 6 months post-treatment, only six patients (9%) had a sustained normalization of ALT. The frequency of the allele DRB1*0404 was significantly higher in patients with a sustained response as compared to those lacking such a response (25.0% vs 2.3%, with a Bonferonni-corrected P-value of 0.019). There was no difference in the frequency of other class II alleles at the DRB1 and DQB1 loci in responders as compared with non-responders. Therefore, we conclude that the maintenance of a response to IFN in chronic hepatitis C may be, in part, determined by genetic factors in the host.

Overexpression of the myristoylated alanine-rich C kinase substrate decreases uptake and K(+)-evoked release of noradrenaline in the human neuroblastoma SH-SY5Y.

The aim of this study was to investigate a possible role of the myristoylated alanine-rich C kinase substrate (MARCKS) in the mechanism of noradrenaline uptake and release in the human neuroblastoma cell line SH-SY5Y. A stable cell line showing a twofold overexpression of MARCKS was prepared by transfecting SH-SY5Y with pCEP4 containing MARCKS cDNA in the sense orientation. This cell line showed no changes in the expression of neurofilaments or markers of noradrenergic large dense-cored vesicles compared with both untransfected SH-SY5Y and SH-SY5Y transfected with pCEP4 only (mock transfected). Similarly, no differences in the rate of cell growth could be detected between these three cell lines. In contrast, specific uptake and depolarization-evoked (100 mM K(+)) release of noradrenaline from the cell line overexpressing MARCKS was inhibited by approximately 50% compared with mock-transfected SH-SY5Y. K(+)-evoked noradrenaline release enhanced by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (100 nM) was also inhibited by 50%. In contrast, carbachol-evoked noradrenaline release was unaffected. Thus, in SH-SY5Y cells, overexpression of MARCKS leads to a decrease in the K(+)-evoked noradrenaline release possibly by increased actin cross-linking preventing the movement of noradrenaline containing large dense-cored vesicles to the plasma membrane in response to depolarization.

Chronic cyclosporin A therapy in rats.

We investigated the pharmacokinetics of cyclosporin A (CsA) blood levels and drug toxicity in a chronic rat study in which long-term (30 weeks) CsA was administered. Ninety Lewis rats received subcutaneous CsA at 5 mg/kg/day for 12 weeks, at which time CsA injections were stopped in 50 animals. The remaining 40 rats were maintained on 5 mg/kg CsA daily until week 18 and then switched to an alternate day dosing until week 30. All rats were observed daily and weighed weekly. Whole blood CsA levels were determined by a commercially available radioimmunoassay kit. The daily dosing regimen resulted in greatly elevated trough CsA levels (greater than 1,600 micrograms/liter) and substantial chronic systemic toxicity, with weight loss and death in eight animals. Alternate day dosing reduced trough levels (mean 1,311 micrograms/liter) and decreased toxicity. Chronic administration by the subcutaneous route resulted in a considerable depot effect, with constancy of drug levels over a 48 hr dosing interval and a slow decline of drug levels (15 days) upon cessation of treatment. These results underscore the importance of monitoring both body weight and blood CsA levels in rodent studies when CsA is employed. Investigators should be aware of drug accumulation with chronic therapy and the consequent need to modify dosing to prevent toxicity.