HLA Family Research Articles

The probability of finding HLA identical or partially matched unrelated donors in the population of Vojvodina

Allogeneic bone marrow transplantation and hematopoietic stem cell transplantation from unrelated donors are treatments of choice for patients lacking HLA identical siblings or family matched donors. Class I HLA typing was performed by using a standard microlymphocytotoxicity test in 434 unrelated persons from Vojvodina, while, class II HLA typing was performed using a modified immunofluorescent technique. The estimated gene frequencies for the populations of Crete, Korea, China, Scotland, Romania, and North America, were used to calculate phenotype frequencies, the probability of finding HLA identical or partially (in 5/6 HLA antigens) matched unrelated donors, the number of donors necessary for research, as well as genetic distances between populations. The probability of finding HLA identical or partially matched unrelated donors for patients from Vojvodina is higher in closely related populations with low genetic distances, such as populations of Crete, Romania and Scotland. The probability of finding HLA identical or partially matched unrelated donors is in inverse proportion with the number of unrelated donors necessary for research with aim of finding at least one HLA compatible donor. The probability of finding compatible unrelated donors depends on the degree of HLA matching between the donor and recipient, HLA phenotype frequencies and the donor pool size. These methodology may have a wider usage, because it can be applied in calculating the probability of finding suitable genotypically matched donors, by using HLA allele frequencies defined by molecular techniques.

Fludarabine Based Conditioning for Allogeneic Transplantation in Severe Aplastic Anemia.

Patients with severe aplastic anemia (SAA) who are multiply transfused or septic have a poor outcome after allogeneic stem cell transplantation. Forty three patients (31 males and 12 females) with SAA underwent allogeneic BMT using a fludarabine based conditioning regimen between 1998 and 2005. The median age was 20 years (range: 4–38) with 11 children and 32 adults. All donors were 6 antigen matched HLA identical sibling or family donors. Co morbidities seen included bacterial sepsis in 15 patients, fungal pneumonia in 4 and a recent intracranial bleed in 5 patients. Seven patients had failed Antithymocyte or antilymphocyte globulin (ATG/ALG) and two patients had failed their first transplant. The median time from diagnosis to transplant was 12 months (range: 2 – 96) and the median transfusions prior to BMT was 28 (range: 2 – 380). Conditioning therapy consisted of: Fludarabine (Flu) 180 mg/m2 over 6d + Busulfan (Bu) 8 mg/kg over 2d + ATG 40 mg/kg/day over 4 d in 17 patients, Flu 180 mg/m2 over 6d + Cyclophosphamide (Cy) 120 mg/kg over 2d ± ATG 40 mg/kg/day over 4d in 17 patients, Flu/TBI/OKT3 in 4, and Cy 120 + Flu 150mg/m2 in 5 patients. Graft versus host disease (GVHD) prophylaxis consisted of Cyclosporine and mini methotrexate. Graft source was peripheral blood stem cells in 39 patients and G-CSF stimulated bone marrow in 4. The median cell dose was 5.2 x 108 MNC/kg (range: 2.1 – 13.6) for PBSC and 5.2 x 108 TNC/kg (range: 3.7 – 6.8) for bone marrow. Five patients expired within the first 10 days due to sepsis. Thirty seven patients engrafted with a mean time to ANC > 500 of 11.6 days (range: 8 – 18) and median platelet engraftment time of 13 days (range: 8 – 32). One patient had primary graft failure and expired on day 64 due to fungal pneumonia despite a second transplant. Acute GVHD was seen in 14 patients (38%) with Grade III–IV GVHD in 4 (10.5%). Chronic GVHD was seen in 10 patients with 6 having limited and 4 with extensive GVHD. Two patients had secondary graft rejection on day + 24 and +60 respectively and expired due to fungal pneumonia. At a median follow up of 17 months (range: 5 – 78); 29 patients (67.7%) are alive and well. Among patients treated with Flu/Bu/ATG, 12/17 (70.5%) are alive and well while the DFS is 82% (14/17) in patients treated with Flu/Cy ± ATG. Comparison with patients conditioned with Cy/ATG during 1990–2004 is given in the table. This comparison suggests that a fludarabine based conditioning regimen may be better, with less rejection, than Cy/ATG for allogeneic BMT in sick patients with SAA who are infected and multiply transfused at the time of BMT.Comparative dataFludarabineCy/ATGNumber4326Rejection3 (7%)7 (27%)DFS29(67.7%)11 (46%)

Human Hepatic Stellate Cells Show Features of Antigen–Presenting Cells and Stimulate Lymphocyte Proliferation

Following cell activation, hepatic stellate cells (HSCs) acquire proinflammatory and profibrogenic properties. We investigated whether activated HSCs also display immune properties. Here we show that cultured human HSCs express membrane proteins involved in antigen presentation, including members of the HLA family (HLA–I and HLA–II), lipid–presenting molecules (CD1b and CD1c), and factors involved in T–cell activation (CD40 and CD80). Exposure of HSCs to proinflammatory cytokines markedly up–regulates these molecules. Importantly, cells freshly isolated from human cirrhotic livers (in vivo activated HSCs) highly express HLA–II and CD40, suggesting that HSCs can act as antigen–presenting cells (APCs) in human fibrogenesis. We also explored whether human HSCs can efficiently process exogenous antigens. Activated HSCs internalize low– and high–molecular–weight dextran and transferrin, indicating that they can perform fluid–phase and receptor–mediated endocytosis. Moreover, HSCs can perform phagocytosis of macromolecules because they internalize latex particles as well as bacteria. Interestingly, both culture–activated and in vivo activated HSCs express high levels of CD68, a protein involved in antigen trafficking. Finally, we studied whether HSCs modulate T–lymphocyte proliferation. In basal conditions, coculture of irradiated HSCs barely induces allogeneic T–lymphocyte proliferation. However, cytokine–stimulated HSCs stimulate the allogeneic T–lymphocyte response in an HLA–II—dependent manner. In conclusion, human activated HSCs express molecules for antigen presentation, internalize macromolecules, and modulate T–lymphocyte proliferation. These results suggest that HSCs may play a role in the immune function of the liver. (Hepatology 2003;38:919-929).

Human hepatic stellate cells show features of antigen-presenting cells and stimulate lymphocyte proliferation

Following cell activation, hepatic stellate cells (HSCs) acquire proinflammatory and profibrogenic properties. We investigated whether activated HSCs also display immune properties. Here we show that cultured human HSCs express membrane proteins involved in antigen presentation, including members of the HLA family (HLA-I and HLA-II), lipid-presenting molecules (CD1b and CD1c), and factors involved in T-cell activation (CD40 and CD80). Exposure of HSCs to proinflammatory cytokines markedly up-regulates these molecules. Importantly, cells freshly isolated from human cirrhotic livers ( in vivo activated HSCs) highly express HLA-II and CD40, suggesting that HSCs can act as antigen-presenting cells (APCs) in human fibrogenesis. We also explored whether human HSCs can efficiently process exogenous antigens. Activated HSCs internalize low- and high-molecular-weight dextran and transferrin, indicating that they can perform fluid-phase and receptor-mediated endocytosis. Moreover, HSCs can perform phagocytosis of macromolecules because they internalize latex particles as well as bacteria. Interestingly, both culture-activated and in vivo activated HSCs express high levels of CD68, a protein involved in antigen trafficking. Finally, we studied whether HSCs modulate T-lymphocyte proliferation. In basal conditions, coculture of irradiated HSCs barely induces allogeneic T-lymphocyte proliferation. However, cytokine-stimulated HSCs stimulate the allogeneic T-lymphocyte response in an HLA-II-dependent manner. In conclusion, human activated HSCs express molecules for antigen presentation, internalize macromolecules, and modulate T-lymphocyte proliferation. These results suggest that HSCs may play a role in the immune function of the liver. (H epatology 2003;38:919-929.)

Familial bipolar disorder and multiple sclerosis: a three-generation HLA family study

The coexistence of bipolar disorder (BD) and multiple sclerosis (MS) is well known. Manic symptoms may represent initial symptoms of MS, at least in some cases, and follow the MS-HLA phenotype frequencies. The purpose of this study was to examine the possible relation of BD and MS based on an HLA family study of a woman with BD and comorbid MS, with family history of BD. Five members of the family from three generations (the patient, her mother, her brother, and her two daughters) were examined regarding the two disorders and the HLA class I and II specificities, performed by serology and molecular techniques. Her deceased father, her brother, and her older daughter suffered from BD. Moreover, in her brother, BD and MS comorbidity was diagnosed. The three affected members and the nonaffected grandmother share the same class I and II, HLA-A2, B18, CW8, DR2, DQ1 haplotype. The shared class II, HLA-DR2, DQ1 haplotype among affected individuals, which is well known to be associated with MS in Caucasians, suggests a possible susceptibility locus for BD, mapped on chromosome 6, very close to the HLA region, underlying the clinical comorbidity of the two disorders.

Myeloma and race: a review of the literature.

Multiple myeloma is an uncommon disease, with approximately 12,000 cases per year diagnosed in America. Blacks have had at least double the risk of being diagnosed with myeloma, and have had twice the mortality rate from the disease compared to whites. Research of the origins of this difference has yielded both insight and controversy. Obesity is likely a risk factor for myeloma, in both blacks and whites. Obesity is more prevalent in the black population, and this may help explain some of the increased incidence of myeloma. Also, genetic factors such as HLA antigens and family history seem to be important in explaining the differential risk of myeloma. Exposure to immunological challenges, especially urinary tract infections in black men, seems important in explaining some of the excess risk in blacks. Factors such as socioeconomic status, dietary preferences, vitamin intake, alcohol and tobacco use, either lack a consensus finding, or may not play a role in explaining the increased myeloma morbidity and mortality in blacks.

HLA family study in a group of Greek women with non-classical 21-hydroxylase deficiency.

HLA family study in a group of Greek women with non-classical 21-hydroxylase deficiency.

The role of marrow grafts in irradiation accidents

In our first paper describing attempted marrow grafts in human beings, my colleagues and I pointed out the potential application of marrow grafting for treatment of irradiation victims (10). Fortunately, in the intervening years irradiation accidents have been infrequent. The only patient clearly to have benefited from a marrow infusion was fortunate enough to have an identical twin (11). The use of marrow grafts after the accident in Yugoslavia and the Chernobyl disaster has attracted a great deal of publicity, but has not clearly benefited any of the recipients (1, 4, 6). Evaluation and supportive care of individuals exposed to potentially lethal doses of total body irradiation have been described in detail (8). In an actual disaster, management is confounded by associated problems of burns and trauma as described following the Chernobyl accident. For this reason and because of problems in assessing total dose and dose distribution, an appropriate application of marrow grafting to irradiation victims is extremely difficult. Studies in mice had suggested that an infusion of allogeneic marrow might actually be harmful if the recipient had received a barely lethal exposure, the so called “midlethal” effect ( 12). This question was studied in the canine model in which the LD-100 dose is approximately 400 cGy. Dogs were exposed to 360, 450, and 540 cGy and given an infusion of allogeneic marrow from unrelated dogs. The hematologic data and the time of death were approximately the same as for dogs given irradiation only. Dogs given autologous marrow infusions at these exposure levels all recovered. It was concluded that an allogeneic marrow infusion from unrelated animals was neither harmful nor beneficial (9). More recently, this same question has been studied in irradiated dogs given an infusion of DLA compatible marrow. Dogs given 450 cGy showed transient engraftment which permitted them to live long enough for their own marrow to recover. At 600 cGy, mixed marrow chimerism was often observed. At increasing irradiation doses of 600,700, and 800 cGy, fewer dogs survived with autologous marrow recovery and more showed sustained allogeneic engraftment (7). These studies demonstrated that an infusion of histocompatible marrow is not harmful and may result in temporary donor marrow function which may prevent lethality until host marrow can recover. At higher lethal exposures, host marrow may not recover and survival depends upon function of the allogeneic graft. It seems clear that to have any chance of success in protection against lethal irradiation, the patient must be histocompatible with the donor, which means that the donor must be syngeneic, autologous, or HLA-identical. Autologous marrow can be cryopreserved for long periods of time (2). The logistics and cost of marrow storage for large numbers of people at risk of irradiation accidents make this approach impractical. A major problem, as emphasized previously (8), concerns histocompatibility typing of the irradiation victim. Blood samples must be obtained promptly after the irradiation exposure before lymphocytes disappear from the circulating blood. When sufficient numbers of viable cells for standard HLA typing are lacking, it may be possible to complete an informative HLA family study by collecting from the patient nucleated cells by marrow aspiration or skin biopsy to provide DNA for restriction fragment-length polymorphism (RFLP) analysis using one or two restriction enzymes and hybridization with class I (HLA-A, B, C) and class II (HLADR, DQ and DP) probes. This test can be used to identify an HLA identical sibling, but will not be adequate for precise matching with unrelated donors. An RFLP test requires 5 to 7 days to complete. Within 1 to 2 years, sufficient numbers of oligonucleotide probes for DR. DQ, and DP genes should be available to allow for typing of DNA amplified by the polymerase chain reaction (PCR) (3). This method requires very small amounts of DNA, a single cell may be an adequate source, and the entire oligo-

Genetic control of in vivo IgE antibody production specific to Dermatophagoides farinae.

HLA population and family study were performed in individuals allergic to Dermatophagoides farinae. Manifestation of allergic symptoms and IgE production to D. farinae are possibly regulated in an autorecessive HLA-linked hereditary manner. This regulation may be focused on a lack of IgE Is gene. IgG and IgG4 antibody production to D. farinae may be regulated in the same manner as symptoms and IgE antibody production.

HLA-TYPING IN MULTIPLE SCLEROSIS SIBLING PAIRS

40 sibling pairs with multiple sclerosis (MS) from 36 families were typed for HLA antigens. The frequency of haplotypic sharing in this population-based group of patients differed little from that expected by chance. The findings are strong evidence against a single MS susceptibility gene closely linked to HLA. This evidence conflicts with previous pooled data which have been considered to prove the existence of such a gene and questions the validity of such data in HLA family studies.

HLA family studies and multiple sclerosis: A common gene, dominantly expressed

All available Australian families with more than one member suffering from multiple sclerosis (MS) were HLA typed. As with all other individual published studies, convincing evidence for linkage between the HLA system and disease was not obtained. An analysis of 100 published affected sib-pairs and 17 cousin-pairs, however, established the existence of an HLA-linked disease susceptibility gene for MS, which is likely to be dominantly expressed. Dominance was also supported by the finding of only three HLA-DR2 (Dw2) homozygous individuals out of 60 unrelated patients which enabled rejection of a recessive gene hypothesis (p < 0.02). Analysis of the sib-pair data strongly suggested that this MS gene is not rare in the normal population and may be as common as DR2.