In vivo T cell depletion with pretransplant anti-thymocyte globulin reduces graft-versus-host disease without increasing relapse in good risk myeloid leukemia patients after stem cell transplantation from matched related donors.

One-hundred and two patients with good risk myeloid leukemia (CML first chronic phase or AML first CR) were transplanted from HLA-related donors after conditioning with (n = 45) or without anti-thymocyte globulin (ATG) (n = 57). One graft failure was observed in the non-ATG and none in the ATG group. The median time to leukocyte engraftment (> 1 x 10(9)/l) was 16 (range 12-33) in the ATG group and 17 days (range 11-29) in the non-ATG group (NS) and for platelet engraftment (> 20 x 10(9)/l) 24 and 19 days (P = 0.002), respectively. Acute GVHD grade II-IV was observed in 47% of the non-ATG and in 20% of the ATG group (P = 0.004). Grade III/IV GVHD occurred in 7% of the ATG and in 32% of the non-ATG group (P = 0.002). Chronic GVHD was seen in 36% and 67% (P = 0.005), respectively. After a median follow-up of 48 months (range 2-128), the 5-year estimated OS is 66% (95% KI: 51-81%) for the ATG group and 59% (95% KI: 46-72%) for the non-ATG group (NS). The 5-year estimated DFS is 64% (95% KI: 50-78%) for ATG and 55% (95% KI: 43-67%) for the non-ATG regimen (NS). The 5-year probability of relapse was 5% in the ATG and 15% in the non-ATG group (NS). ATG as part of the conditioning regimen leads to a significant reduction in GVHD without increase of relapse in patients with myeloid leukemia after stem cell transplantation from HLA-related donors.

Patient cytomegalovirus seropositivity with or without reactivation is the most important prognostic factor for survival and treatment-related mortality in stem cell transplantation from unrelated donors using pretransplant in vivo T-cell depletion with anti-thymocyte globulin.

We evaluated the cytomegalovirus (CMV) serostatus as a risk factor for survival and treatment-related mortality (TRM) in 125 patients allografted from an unrelated donor between 1994 and 1999. All patients received pretransplant in vivo T-cell depletion using rabbit anti-thymocyte globulin (ATG). Only one patient had primary graft failure and severe grade III/IV graft-versus-host disease occurred in 14% of the patients. The overall survival (OS) at 3 years was 70% for CMV-negative patients (n = 76) and 29% in the seropositive cohort (n = 49) (P > 0.001). In multivariate analyses, CMV seropositivity remained an independent negative prognostic factor for OS (RR: 2.1; CI: 1.2-3.8; P = 0.014), apart from age > 20 years (RR: 2.74; CI: 1.2-3.8; P = 0.004) and late leucocyte engraftment (RR: 2.4; CI: 1.2-4.9; P = 0.015). The TRM for all patients was 27%. Despite monitoring for CMV antigenaemia and preemptive therapy with ganciclovir when reactivation occurred, seropositive patients had a three times higher risk of fatal treatment-related complications than seronegative patients. In multivariate analyses, CMV seropositivity remained the strongest independent negative factor for TRM (RR: 5.3; CI: 1.9-14.6; P = 0.002), followed by age > 20 years (RR: 4.8; CI: 1.3-18.1; P = 0.02) and delayed leucocyte engraftment (RR: 3.6; CI: 1.2-11; P = 0.02). The TRM was identical in seropositive patients with (n = 27) or without (n = 22) CMV reactivation (44% versus 50%). We conclude that CMV seropositivity, despite preemptive ganciclovir therapy and even without reactivation, is a major negative prognostic factor for survival as well as for TRM in unrelated stem cell transplantation using pretransplant in vivo T-cell depletion with ATG.

Anti-thymocyte-globulin as part of the preparative regimen prevents graft failure and severe graft versus host disease (GvHD) in allogeneic stem cell transplantation from unrelated donors.

To reduce the incidence of GvHD and the rate of graft failure in unrelated stem cell transplantation, we incorporate anti-thymocyte globulin in the preparative regimen in 98 patients with hematological or inherited storage disease. The median age was 32 years (range: 1-56) and 84 patients underwent transplantation from HLA-A,-B and DR identical donor, while in 14 patients the donor were mismatched either in HLA- A, -B or -DR locus. Only one patient with chronic myelocytic leukemia (CML) and blast crisis had a primary graft failure (1%). Grade II-IV acute GvHD occurred in 37 patients (37%), grade III/IV GvHD developed in 15 patients (15%). Chronic GvHD was observed in 29%, and only 12 patients had extensive GvHD (17%). After a median follow-up of 34 months (range, 9-90), the estimated overall survival at 3 years for all patients is 58% (CI 95%: 48%-68%), and the estimated disease-free survival at 3 years is 49% (CI 95%: 38%-60%). For patients with CML transplanted in first chronic phase or accelerated phase (n=40), the estimated overall survival at 3 years is 70% (CI 95%: 56%-84%), and the estimated disease-free survival at 3 years is 58% (CI 95%: 17%-85%). ATG in unrelated stem cell transplantation reduces the risk of severe acute and chronic GvHD and of graft failure without an obvious increase of severe infection. Further follow-up is mandatory to determine the incidence of late relapse.

Comparison of total body irradiation vs busulfan in combination with cyclophosphamide as conditioning for unrelated stem cell transplantation in CML patients.

We compared fractionated total body irradiation (12 Gy)/cyclophosphamide (120 mg/kg) with busulfan (16 mg/kg)/cyclophosphamide (120 mg/kg) as preparative therapy in unrelated donor stem cell transplantation of CML patients. Fifty patients with CML (1.CP = 46; aP = 4) and a median age of 36 years (range 16-52) were enrolled in this sequential trial between 1994 and 1999. In both groups patients were well balanced with respect to age, disease status, stem cell source and CMV status. All patients received standard doses of cyclosporin A, methotrexate and anti-thymocyte globulin (ATG) as GVHD prophylaxis. No graft failures occurred in either group. The median day of leukocyte engraftment was earlier in the Bu/Cy than in the TBI/Cy group (day 15 vs 17; P = 0.006). The incidence of grade II-IV GVHD was 40% in the TBI/Cy and 36% in the Bu/Cy group, whereas severe grade III/IV GVHD was only observed in 12% of patients in both groups. The incidence of chronic GVHD (limited and extensive) at 1 year was higher in the Bu/Cy arm (65% vs 30%; P = 0.02). More toxicity grade I/II of the liver (88% vs 44%; P = 0.002) and more hemorrhagic cystitis (32% vs 8%; P = 0.02) were observed in the Bu/Cy regimen. Seven relapses in the TBI and no relapse in the Bu/Cy group were observed after a median follow-up of 44 and 15 months, respectively. The estimated 3 year OS and DFS was 72% (95% CI: 55-98%) and 58% (95% CI: 39-77%) in the TBI and 70% (95% CI: 51-89%) for DFS and OS in the Bu/Cy group. We conclude that the anti-leukemic effect of the Bu/Cy regimen seems to be at least as effective as the TBI/Cy combination in unrelated stem cell transplantation of CML patients, with no graft failures, but that it correlates with a higher incidence of liver toxicity, hemorrhagic cystitis and chronic GVHD. Longer follow-up is necessary to determine the late relapse rate and late toxicity.

A randomized comparison of once versus twice daily recombinant human granulocyte colony-stimulating factor (filgrastim) for stem cell mobilization in healthy donors for allogeneic transplantation.

To evaluate the schedule dependency of granulocyte colony-stimulating factor (G-CSF) (filgrastim) for stem cell mobilization, we conducted a randomized comparison in 50 healthy donors, with one subcutaneous daily injection of 10 microg/kg G-CSF (n = 25) compared with twice injections daily of 5 microg/kg G-CSF (n = 25). The two groups were well balanced for age, body weight and sex. G-CSF application was performed on an out-patient basis and leukapheresis was started in all donors on day 5. The most frequent side-effects of G-CSF were mild to moderate bone pain (88%), mild headache (72%), mild fatigue (48-60%) and nausea (8%) without differences between the two groups. The CD34(+) cell count in the first apheresis was 5.4 x 10(6)/kg donor weight (range 2.8-13.3) in the 2 x 5 microg/kg group compared with 4.0 x 10(6)/kg (range 0.4-8.8) in the 1 x 10 microg/kg group (P = 0.007). The target of collecting > 3.0 x 10(6) CD34(+) cells/kg donor weight with one apheresis procedure was achieved in 24/25 (96%) donors in the 2 x 5 microg/kg group and in 17/25 (68%) donors in the 1 x 10 microg/kg group. The target of collecting > 5.0 x 10(6) CD34(+) cells/kg in the first apheresis was achieved in 64% in the 2 x 5 microg/kg group, but in only 36% in the 1 x 10 microg/kg group. The progenitor cell assay for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) was higher in the 2 x 5 microg/kg group than in the 1 x 10 microg/kg group (7.0 vs. 3.5 x 10(5)/kg, P = 0.01; 6.6 vs. 5.0 x 10(5)/kg; P = 0.1). Administering G-CSF (filgrastim) at a dosage of 5 microg/kg twice daily rather than 10 microg/kg once daily is recommended; this leads to a higher CD34(+) cell yield and requires fewer apheresis procedures without increasing toxicity or cost.

Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by T(h)3/T(r)1-type cytokines IL-10 and transforming growth factor-beta but not by a T(h)1 to T(h)2 shift.

Exposure to infective larvae of the filarial nematode Onchocerca volvulus (Ov) either results in patent infection (microfilaridermia) or it leads to a status called putative immunity, characterized by resistance to infection. Similar to other chronic helminth infections, there is a T cell proliferative hyporesponsiveness to Ov antigen (OvAg) by peripheral blood mononuclear cells (PBMC) from individuals with patent infection, i.e. generalized onchocerciasis (GEO), compared to PBMC from putatively immune (PI) individuals. In this study, mechanisms mediating this cellular hyporesponsiveness in GEO were investigated: the low proliferative response in PBMC from GEO individuals was associated with a lack of IL-4 production and significantly lower production of IL-5 compared to those from PI individuals, arguing against a general shift towards a T(h)2 response being the cause of hyporesponsiveness. In contrast, IL-10 and transforming growth factor (TGF)-beta, two cytokines associated with a T(h)3 response, seemed to mediate hyporesponsiveness: PBMC from individuals with GEO produced significantly more IL-10, and T cell proliferative hyporesponsiveness in this group could be reversed by the addition of anti-IL-10 and anti-TGF-beta antibodies. Hyporesponsiveness was specific for OvAg and not observed upon stimulation with related nematode antigens, arguing for a T cell-mediated, Ov-specific down-regulation. Ov-specific T cells could be cloned from GEO PBMC which have a unique cytokine profile (no IL-2 but high IL-10 and/or TGF-beta production), similar to the T cell subsets known to suppress ongoing inflammation (T(h)3 and T(r)1), indicating that this cell type which has not been found so far in infectious diseases may be involved in maintaining Ov-specific hyporesponsiveness.

Comparison of progenitor cell collection on day 4 or day 5 after steady-state stimulation with G-CSF alone in breast cancer patients: influence on CD34+ cell yield, subpopulation, and breast cancer cell contamination.

To determine the influence of apheresis timing on CD34+ cell yield, subpopulation, and breast cancer cell contamination, 48 women with breast cancer were stimulated from steady-state hematopoiesis in a prospective but nonrandomized study with 2 x 5 microg/kg G-CSF s.c. alone, and apheresis was started either on day 4 (n = 24) or day 5 (n = 24). Forty-eight women with breast cancer (stage II/III, n = 30; stage IV; n = 12; inflammatory, n = 6) and a median age of 44 years were well balanced between the two groups. In group I, aphersis was started on day 4 and additionally performed on day 5 after G-CSF stimulation, and in group II, apheresis was started on day 5. CD34+ cell count and CD34+ cell subpopulation were determined according to international criteria. Breast cancer cell contamination was detected by immunocytology. The median CD34+ cell harvest on day 4 was 3.3 x 10(6)/kg body weight (range 0.5-12.8) and 6 x 10(6)/kg BW (range 0.3-30) for patients starting on day 5 (p = 0.01). Those patients starting on day 4 achieved a median CD34+ cell count of 4 x 10(6)/kg (range 0.7-13) on day 5 (NS). Twenty-one percent of group I and 71% of group II achieved >5 x 10(6)/kg BW CD34+ cells in the first apheresis, whereas <2.5 x 10(6)/kg BW CD34+ cells in the first apheresis were observed in 38% of group I and 16% of group II. No differences were observed between the CD34+ cell subpopulations, CD34+/CD38+ (10.5% versus 10.5%) and CD34+/Thyl+ (1.5% versus 1.8%). The CD34+ cell harvest from consecutive collecting on days 4 and 5 was nearly identical to the harvest starting on day 5 (6.4 versus 6 x 10(6)/kg). Collecting CD34+ progenitor cells after stimulation with G-CSF alone on day 5 results in a significantly higher cell yield than starting collecting on day 4. No differences in respect to breast cancer cell contamination and CD34+ cell subpopulation were observed.

Efficacy of further attempts to mobilize CD34+ peripheral stem cells with alternative procedures after primary failure.

19 patients who failed the target collection of at least 2.5 x 10(6) CD34+ cells/kg underwent further mobilization procedures either with granulocyte-colony-stimulating factor (G-CSF) alone after failure to chemotherapy plus G-CSF (group 1), or with chemotherapy plus G-CSF (group 2), or with high-dose G-CSF (24 microg/kg) alone (group 3) after failure to respond to standard dose of G-CSF (10 microg/kg) alone. In all groups, an increase in median CD34+ cell yield could be observed following alternative procedures (1.1- to 1.9 x 10(6) kg; p = 0.02). The highest increase in CD34+ cell harvest was achieved in group 1 (0.85 to 2.2 x 10(6) kg), followed by group 2 (1. 2 to 1.7) and group 3 (1.0 to 1.4), but without statistically significant difference between the mobilization technologies. All patients with more than 1.0 x 10(6) CD34+ cells/kg in the first apheresis procedure reached the overall target of 2.5 x 10(6) CD34+ cells/kg after a second or subsequent mobilization procedure. In contrast, only 3 of 8 patients (37%) with less than 1.0 x 10(6) CD34+ cells in the first harvest could reach the target of 2.5 x 10(6) CD34+ cells after further mobilization attempts.

[Structure of a cell bank for transplantation of HLA-typed, cryopreserved human adult retinal pigment epithelial cells]. / Aufbau einer Zellbank für die Transplantation von HLA-typisierten, kryokonservierten humanen adulten retinalen Pigmentepithelzellen.

BACKGROUND: The transplantation of retinal pigment epithelial cells (RPE) in patients with age-related macular degeneration is discussed as a future therapy. A cornea bank can serve as a source for cells that can be isolated, cultivated, HLA-typed and cryopreserved for subsequent tissue-compatible transplantation. METHODS: RPE cells are isolated enzymatically from donor eyes and are cultured in a specially designed growth medium. After multiplication, one part of the culture is cryopreserved; the other part is subcultured for HLA-typing. Completely typed and morphologically sufficiently well-differentiated cell cultures are registered on a donor list (RPE cell bank) and can be provided for cell transplantations with matching HLA type in patients suffering from RPE degenerative diseases. RESULTS: A total of 461 cell cultures have been prepared since 1996; 116 fully typed and well-differentiated cell cultures are stored in our cell bank. Since January 1998 patients who agreed to have an RPE transplantation have been registered on a waiting list. Seven transplantations have already been performed. CONCLUSION: RPE cells can be stored cryopreserved in a cell bank and can be kept available for transplantation for a prolonged period of time.

Improved outcome in haemophagocytic lymphohistiocytosis after bone marrow transplantation from related and unrelated donors: a single-centre experience of 12 patients.

Haemophagocytic lymphohistiocytosis (HLH) is an autosomal recessive disease with histiocytic and lymphocytic infiltrations in multiple organs. Cure seems possible only by allogeneic bone marrow transplantation (BMT), but matched sibling donors (MSD) are restricted and high mortality rates are associated with BMT from unrelated donors (URD). We report on 12 consecutive HLH patients with an improved outcome following URD transplants. Eight patients received BMT from URD, four from MSD. Five patients had signs of active HLH at the time of BMT. The conditioning regimen consisted of 20 mg/kg busulphan, 60 mg/kg VP-16 and 120 mg/kg cyclophosphamide and, in case of URD, 90 mg/kg antithymocyte globulin. The doses of busulphan and VP-16 were reduced during the programme to 16 mg/kg and 30 mg/kg, respectively. Using a fivefold graft-versus-host disease (GVHD) prophylaxis, GVHD was absent or mild in 10, and moderate or severe in two patients undergoing unrelated transplants. One patient with URD experienced graft failure and was retransplanted on day 37. Major toxicities were hepatic veno-occlusive disease in five, capillary leak syndrome in two, pneumonia in three, sepsis in one, severe mucositis in one and seizures in two patients. All patients are alive without HLH after a median follow-up of 24.5 months. One patient has chronic GVHD, another patient has severe retardation. Three patients show slight to moderate development delay. These results indicate that in HLH, BMT from matched unrelated donors should be performed. Incomplete resolution of disease activity need not impede a successful outcome.

[Natural human IgM-antibodies in neuroblastoma therapy: preliminary findings of a phase I/II clinical trial]. / Natürliche humane IgM-Antikörper in der Therapie des Neuroblastoms: Vorläufige Ergebnisse einer Phase I/II-Therapiestudie.

BACKGROUND: Sera from healthy individuals contain natural IgM antibodies (anti-NB-Ab) cytotoxic to neuroblastoma (NB) cells. In contrast to healthy children the prevalence of anti-NB-Ab in sera of NB patients is low. Binding of anti-NB-Ab to the NB cell surface leads to activation of complement in vitro. In vivo the injection of the purified IgM fraction from a cytotoxic blood donor results in complete growth arrest of NB xenografts in nude rats. Preliminary results from a phase I/II study to evaluate the pharmacokinetics and side effects of a therapy with anti-NB-Ab are presented here. PATIENTS: Included in this study are patients with NB stage 4 according to INSS with relapse or non-responders to therapy according to the GPOH-NB-therapy protocol. The patients are negative for anti-NB-Ab and are older than one year. METHODS: The therapy is based on a complete exchange of the anti-NB-Ab negative patient serum against serum from an anti-NB-Ab positive ABO-compatible donor by means of plasmapheresis. RESULTS: Up to now, 14 cycles of plasmapheresis have been carried out in 6 NB patients. In 13 of 14 therapy cycles a significant increase in serum toxicity could be observed. Severe side effects have not been seen except a catheter associated thrombosis which was reversible under heparin treatment. After plasmapheresis, pain in the tumor site or regions of metastasis did occur regularly. In some cases temporary elevation of body temperature occurred. One patient had a reduced MIBG uptake after therapy. Tumor necrosis was observed in 2 patients. Three patients showed tumor progress. CONCLUSION: Immunotherapy of NB in children by serum exchange using anti-NB-Ab positive ABO-compatible donor serum is feasible without major side effects and leads to a significant increase of serum toxicity against NB cells.

Correlation of thymic pathology with HLA in myasthenia gravis.

The aim of this study was to investigate associations between thymic pathology and HLA in myasthenia gravis. HLA typing was performed in 95 of 125 Caucasian patients who underwent transsternal thymectomy for myasthenia gravis between 1976 and 1995. Multiple comparison procedures applied within each HLA locus demonstrated significant correlations between the ancestral suprahaplotype A1 B8 DRB1*0301 DRB3*0101 DQA1*0501 and thymic hyperplasia and between HLA-A24 and thymoma. A weaker association was found between A3 and thymic atrophy and thymolipoma. On logistic discriminant analysis, HLA-B8 (P = 0.001) and HLA-A3 (P = 0.028) were identified as the only significant classifiers to jointly provide a good discriminator between the thymic pathologies. When the suitability of HLA for detection of thymoma was examined in a second logistic regression analysis, both HLA-A24 (OR 9.7; 95% CI [1.6, 73.7]) and HLA-B8 (OR 0.1; 95% CI [0.0, 0.5]) were significant predictive factors. The above correlations between thymic pathology and HLA-A3, HLA-A24, and HLA-B8 (but not MHC class II alleles) suggest an involvement of MHC class I restricted T cells in myasthenic autoimmunity that may partially be reflected by thymic pathology.

Use of a five-agent GVHD prevention regimen in recipients of unrelated donor marrow.

A five-agent GVHD prophylaxis programme consisting of cyclosporin A, methotrexate, anti-thymocyte-globulin, pentaglobin and metronidazol was given to 48 recipients of unrelated donor marrow with chronic myelogenous leukemia, acute leukemia, myelodysplastic syndromes, and familiar lymphocytic hemophagocytosis of an average age of 33.5 (0.6-56) years. GVHD grades II-IV occurred in 18 patients (39%) and grades III-IV in five patients (11%). Chronic GVHD developed in nine patients (23%), three limited and six extensive. Fifteen patients died. Clinical relapse was detected in eight patients. Four patients died as a consequence of the underlying disease and subsequent treatment, 11 patients died of transplant-related causes. After a median follow-up of 19 months, the overall and disease-free survival are 67% and 62%, respectively. Survival by age is as follows: 0-19 years: 12/13 patients; 20-39 years: 14/25 patients; 40-59 years: 7/10 patients. The five-agent GVHD prophylaxis regimen is effective. Matched-unrelated donor transplants can be carried out safely in patients younger than 50 years of age. The results in patients younger than 20 years of age should encourage matched-unrelated donor transplants at earlier stages of the disease.

Stem cell mobilization with G-CSF alone in breast cancer patients: higher progenitor cell yield by delivering divided doses (2 x 5 microg/kg) compared to a single dose (1 x 10 microg/kg).

We investigated the schedule dependency of G-CSF (10 microg/kg) alone in mobilizing peripheral blood progenitor cells (PBPC) in breast cancer patients. After a median of three cycles (range, 2-6) of anthracycline-based chemotherapy, 49 patients with breast cancer (stage II/III, > or = 10+ Ln n = 36; locally advanced/inflammatory n = 8, stage IV (NED) n = 5) underwent PBPC collection after steady-state mobilization either with 1 x 10 microg/kg (n = 27) or with 2 x 5 microg/kg (n = 22) G-CSF daily for 4 consecutive days until completion of apheresis. Apheresis was started on day 5. Priming with 2 x 5 microg/kg resulted in a higher median number of CD34+ cells (5.8 vs 1.9 x 10(6)/kg, P = 0.003), MNC (6.6 vs 2.6 x 10(8)/kg, P < 0.001) and CFU-GM (6.5 vs 1.3 x 10(4)/kg, P = 0.001) in the first apheresis than with 1 x 10 microg/kg. Also the overall number of collected BFU-E was higher in the 2 x 5 microg group (9.2 vs 3.1 x 10(4)/kg; P = 0.01). After high-dose chemotherapy with cyclophosphamide/thiotepa/mitoxantrone (n = 46) hematopoietic engraftment with leukocyte count > 1.0/nl was reached in both groups after a median of 10 days (range, 8-15) and with platelets count > 50/nl after 12 (range, 9-40) and 13 days (range, 12-41), respectively. A threshold of > 2.5 x 10(6)/kg reinfused CD34+ cells ensured rapid platelet engraftment (12 vs 17 days; P = 0.12). Therefore, the target of collecting > 2.5 x 10(6) CD34+ cells was achieved in 21/27 (80%) patients of the 1 x 10 microg group and in 21/22 (95%) patients of the 2 x 5 microg/kg group with a median of two aphereses (range, 1-4). None in the 10 microg/kg group, but 6/22 (28%) patients in the 2 x 5 microg/kg group required only one apheresis procedure, resulting in fewer apheresis procedures in the 2 x 5 microg/kg group (mean, 1.8 vs 2.3, P = 0.01). These results demonstrate that priming with 10 microg/kg G-CSF alone is well tolerated and effective in mobilizing sufficient numbers of CD34+ cells in breast cancer patients and provide prompt engraftment after CTM high-dose chemotherapy. G-CSF given 5 microg/kg twice daily (2 x 5 microg) leads to a higher harvest of CD34+ cells and required fewer apheresis procedures than when given 10 microg/kg once daily (1 x 10 microg).

Physiological features of primary cultures and subcultures of human retinal pigment epithelial cells before and after cryopreservation for cell transplantation.

BACKGROUND: One striking disadvantage of in vitro culturing of human retinal pigment epithelial (RPE) cells is the loss of epithelial differentiation and specific cell function during culture. This may be one of the main reasons for the failure of RPE cell transplantation. The aim of this study was to evaluate cell culture conditions ensuring the maintenance of differentiation and function of RPE cells after subcultivation and storage in liquid nitrogen. METHODS: Enzymatically isolated cells were seeded onto coated culture dishes, cultured with a specially formulated improved growth medium until confluence and then cryopreserved in liquid nitrogen for 16-66 months. HLA class I and II typing was performed before cryopreservation and after thawing. Expression of Ca2+ channels in primary, first-passage and cryopreserved RPE cells was studied using the patch-clamp technique. RESULTS: After cryopreservation no loss of any HLA antigen was detectable in 12 of 14 cell strains studied. Patch-clamp experiments demonstrated that high-threshold L-type Ca2+ channels, which are typical for freshly isolated cells, could be detected in first-passage and cryopreserved RPE cells only when improved culture conditions were employed, not in conventionally cultured cells. The characteristics of these channels showed little change in subcultured cells compared to primary cultures. CONCLUSION: This is the first study showing the maintenance of adult human RPE-specific cell differentiation and characteristics in vitro after primary culture and after cryopreservation using improved cell culture methods. The optimization and quality control of cell culture is an important prerequisite for successful cell transplantation.

HLA DPA1/DPB1 genotype and haplotype frequencies, and linkage disequilibria in Nigeria, Liberia, and Gabon.

The frequencies of DPA1 and DPB1 alleles and their occurrence in haplotypic linkage were assessed and compared in Nigerian, Liberian, and Gabonese individuals. Differences were seen in the distribution patterns; these differences were more pronounced between the Gabonese and the other two populations than between Liberians and Nigerians. Several haplotypic DPA1-DPB1 combinations could be verified by homozygosity. Linkage disequilibria of DPA1-DPB1 combinations, indicating further probable haplotypes, were estimated. Although different allele and haplotype frequencies were recognized in the three subgroups, the linkage disequilibria were mostly either positive or negative in all populations.

The impact of HLA on long-term outcome after thymectomy for myasthenia gravis.

In a retrospective series of 86 patients with myasthenia gravis, the only factors predictive of improvement in muscular strength after transsternal thymectomy were preoperative severity of myasthenia (90% versus 54%, p = 0.0014) and HLA-B8 (79% versus 50%, p = 0.0060) in bivariable and multivariable analyses. Both factors were not interrelated (p = 0.824). The statistical effect of HLA-B8 was independent from preoperative severity of disease. Typing for HLA-B8 may thus be a valuable adjunct in predicting the benefit of thymectomy in myasthenia. The observation that an MHC class I allele is associated with clinical improvement after thymectomy suggests that the clinical course of myasthenia may be influenced by class I restricted T-cells.

HLA-DP control of human Schistosoma haematobium infection.

The DPA1 and DPB1 alleles of the major histocompatibility complex (MHC) class II were determined in 110 patients and 120 healthy controls of a Gabonese population from an area endemic for Schistosoma haematobium infection. The MHC-DP alleles of the variable second exons and their human leukocyte antigen (HLA) epitopes were correlated with egg excretion, interleukin-4 and interferon-gamma patterns, and bladder abnormalities, as detected by ultrasonography. A methionine at position 11 of the DP alpha molecule (Met-11) and DPA1*0301 were associated with schistosomiasis when compared with controls (phenotypic gene frequencies = 0.791 versus 0.583 and 0.555 versus 0.375, respectively). Met-11 homozygosity occurred more often in patients, whereas healthy controls were more frequently homozygous for an alanine at position 11 (Ala-11). The combination of the DPB1-epitope DEAV (positions 84-87 of the DP beta molecule) and Met-11 positive DPA1 alleles was more frequent in patients than in controls (0.573 versus 0.316). Two years after antischistosomal treatment, the rate of reinfection as examined in 55 of the 110 former patients was higher in DPA1*0301-positive individuals than in those not possessing this allele (P < 0.001). Ala-11 positive individuals showed less frequently ultrasonographic signs of bladder pathology than Ala-11 negative individuals (P < 0.05). Our results suggest a role of MHC-DP elements in the manifestation of disease in S. haematobium infection.

German consensus on immunogenetic donor search for transplantation of allogeneic bone marrow and peripheral blood stem cells.

In Germany allotransplantation of bone marrow or peripheral blood stem cells is presently performed by 34 different teams operating more or less independently. Thus, strategies of immunogenetic donor search, use of the various tissue typing techniques and policy on acceptable HLA mismatches in related and unrelated settings may vary considerably from one transplant centre to another. This paper summarises the results of the first German consensus meeting on immunogenetic donor search for bone marrow/peripheral blood stem cell grafting. The main goal of the participating transplant physicians and immunogeneticists was to define national standards for the above issues.