Ebbe Dickmeiss

Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D☆

A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively.

Effect of thapsigargin on cytoplasmic Ca2+ and proliferation of human lymphocytes in relation to AIDS

The tumor-promoting sesquiterpene lactone, thapsigargin, induced a dose-dependent increase of the cytoplasmic Ca2+ concentration ([ Ca2+]i) in human lymphocytes from a resting level between 100 and 150 nM up to about 1 microM. Half-maximum response was found at about 1 nM of thapsigargin, full response at 100 nM. The effect of thapsigargin on [Ca2+]i exceeded that of phytohaemagglutinin (PHA) which raised [Ca2+]i to maximum 300 nM. In combination with phorbol 12-myristate 13-acetate (PMA), thapsigargin stimulated the proliferation of normal lymphocytes to the same extent as did PHA, whereas the thapsigargin/PMA treatment could not restore the defective proliferation of AIDS lymphocytes in spite of the increased [Ca2+]i. Thapsigargin or PMA added separately had no stimulatory effects on cell proliferation. The thapsigargin/PMA treatment caused an increase in the interleukin-2 (IL-2) production of the lymphocytes, which was much higher than that caused by the PHA treatment, even in AIDS lymphocytes. Moreover, the thapsigargin/PMA treatment stimulated the expression of the IL-2 receptors on both normal and AIDS lymphocytes, similar to the effect of PHA. It is concluded that thapsigargin exerts its effects on lymphocyte proliferation by increasing [Ca2+]i, and that the general defect of AIDS lymphocytes, rather than being ascribed to the initiating signal systems, is associated with later events related to DNA synthesis and proliferation.

Transfusion policy in ABO-incompatible allogeneic stem cell transplantation

As a result of the fact that the human leucocyte antigen (HLA) system is inherited independently from the ABOblood group system, approximately 40–50% of all haematopoietic stem cell transplants (HSCT) are performed across the ABO-blood group barrier. There are three kinds of ABO incompatibility: minor incompatibility (in 20–25% of transplants) is characterized by the ability of donor B lymphocytes to produce antirecipient alloantibodies (e.g. group O donor to a group A recipient). In contrast, major incompatibility (in 20–25% of transplants) is characterized by the presence of antidonor alloantibodies (e.g. group A donor to a group O recipient). Bidirectional ABO-incompatibility (up to 5% of transplants) occurs when both donor and recipient produce alloantibodies against each other’s red cell (e.g. group A donor to a group B recipient). Several studies demonstrated that an ABO mismatch does not affect the incidence of graft rejection and does not cause slower engraftment of neutrophils and platelets. However, haemolytic transfusion reactions may occur [1]. A distinction can be made between immediate (during graft infusion) and delayed (during engraftment) immune haemolysis. Another phenomenon that may occur is pure red cell aplasia (PRCA) which is seen in 15–20% of cases after a major ABO-incompatible transplant. This phenomenon is explained as to be due to alloantibody-producing plasma cells which are relatively resistant to chemoand radiotherapy. Antibodies produced by such surviving plasma cells of the recipient are responsible for the inhibition of the growth of RBC precursors in the bone marrow [2]. Recommendations for transfusion policy in cases of ABO-incompatible stem cell transplantation and management of RBC transfusions in cases of PRCA or relapse of the underlying disease are not well defined in the literature. To obtain information on these aspects the following questions were sent to various transplant centres and departments of blood group serology and transfusion medicine. We received 10 contributions for this Forum. The participating centers are listed in Table 1. In Table 2, all answers are summarized.

HTLV-III Antibody Testing in Three Danish Blood Banks

Abstract. The Organon Teknika Vironostika anti‐HTLV‐III/LAV test was evaluated in three Danish blood banks. The evaluation comprised in total 3,940 consecutive donors. In all three blood banks the tests were carried out exactly according to the manufacturers instructions, using a low cut‐off value defined as (4N̄ + P̄)/5, where N̄ and P̄ are means of optical densities of known negative and positive samples. By this method the overall frequency of repeatably positive samples was 0.30%. When tested by Western blot, however, none of these samples were shown to contain specific antibodies against HTLV‐III/LAV proteins. When testing different categories of patients, only sera containing HLA antibodies gave rise to false‐positive reactions. Finally, important differences in the results were observed regarding sample preparation, single or dual wavelength optical density readings, and the experience of the technical staff.

A comparative study of CD34+ cells, CD34+ subsets, colony forming cells and cobblestone area forming cells in cord blood and bone marrow allografts

Abstract:  Cord blood (CB) has become an alternative source of hematopoietic progenitor cells (HPCs) for allogeneic transplantation. We have developed a new efficient protocol for CB collection. Using this method an average of 17.7 times 108 [range (6.8–29.6) × 108, n = 13] total nucleated cells (TNCs) were harvested. Based on recent Eurocord data, which have shown safe engraftment using a threshold dose of 0.37 times 108 CB TNCs/kg body weight (BW), we calculated that six out of thirteen CB grafts collected by this method were sufficient to engraft adults. The CB derived CD34+ population contained two‐fold higher numbers of committed HPCs (CFU‐GM, BFU‐E) and six‐fold higher numbers of pluripotent HPCs [CD34+/CD38− cells, wk 5 and wk 8 cobblestone area forming cells (CAFCs)] than the CD34+ population of BM. Extrapolation revealed that BM grafts providing the threshold dose for allogeneic transplantation of 2 times 108 TNCs/kg BW contained nearly 3 times more pluripotent HPCs than CB grafts providing the Eurocord threshold dose. The assessment of CD34+/CD38− cell numbers in CB grafts was highly reproducible and correlated well with the in vitro performance of pluripotent HPCs, i.e. numbers of CAFCs. We conclude that CB grafts providing high numbers of TNCs have the potential to engraft adults and that the enumeration of pluripotent HPCs by flow cytometry may be a useful tool to define the ultimate threshold dose for CB transplantation.

The Immunological and Clinical Outcome of HIV Infection: 31 Months of Follow-up in a Cohort of Homosexual Men

T-cell subsets, antibodies (Ab) against human immunodeficiency virus (HIV) and clinical status were evaluated during a 31 (24-35) month follow-up study of homosexual men. The study group included 50 homosexual men, with many sexual partners, who by 1982-83 were without symptoms and had a prevalence of HIV Ab of 38%. Among the men who were seropositive on the initial investigation a significant decrease occurred in the absolute number of CD4+ lymphocytes (p less than 0.01). 88% of these men experienced a decrease, and by follow-up 59% had CD4+ lymphocytes below the normal range. Also the men who seroconverted during the study had a significant decrease in CD4+ lymphocytes, while no changes were observed in the seronegative group. None of the subgroups had significant changes in CD8+ lymphocyte number. AIDS or AIDS related complex developed in 33% of the men seropositive at inclusion. None of these clinical syndromes developed in the seroconverting or the seronegative group. The men who eventually developed clinical symptoms did not differ significantly from the healthy HIV Ab positive persons, with respect to lifestyle parameters, presence of lymphadenopathy and isolation of cytomegalovirus. However, they had significantly lower CD4+ cells and CD4/CD8 ratio (p less than 0.01) at inclusion. It is concluded that in the majority of persons infected with HIV, phenotypic T-cell alterations will occur with a latency of years, but it remains to be seen if the alterations necessarily will result in clinical manifestations. Further, T-cell subset determination among healthy HIV Ab positive persons will provide prognostic information.

Antibodies to human T‐cell lymphotropic virus type III in promiscuous healthy homosexual men. Relation to immunological and clinical findings

Abstract. Antibodies to human T‐cell lymphotropic virus type III (HTLV‐III Ab) were present in twenty‐one out of sixty‐four asymptomatic promiscuous homosexual men from Copenhagen. The presence of HTLV‐III Ab was associated with lymphadenopathy (P<0·0005), cytomegalovirus isolation (P<0·01), low skin test reactivity (P<0·01) and episodes of fever within the 2 month period prior to investigation (P<0·05). No significant differences occurred in the total number of T‐cells, T‐suppressor cytotoxic cells, T‐helper cells or helper to suppressor ratio (H/S ratio) between HTLV‐III Ab positive and negative homosexuals. An H/S ratio ≤1·0 was significantly more frequent in homosexual men who both had HTLV‐III Ab and excreted cytomegalovirus (P<0·01).

Preparation of granulocyte concentrates by apheresis

Bacterial and fungal infections still are life-threatening complications in immunocompromised patients. Transfusion of allogeneic granulocytes, collected by donor-apheresis, may be indicated to control these infections [1]. Sedimentation agents such as highor low molecular weight hydroxyethyl starch (HES) are routinely used to improve the separation of WBCs from RBCs. Administration of steroids (i) cytokines (ii) (recombinant G-CSF) or both (iii) to the donors increases the number of circulating neutrophils in the peripheral blood. This practice allows the collection of about 1 · 10 (i), 5 · 10 (ii) or 8 · 10 (iii) granulocytes per run [2,3]. Granulocyte concentrates still contain significant numbers of red cells and therefore an ABO RhD match with the recipient is mandatory, but they may be transfused across the ABO blood group barrier after red cell and ⁄ or plasma depletion [2]. The modalities of collection of granulocytes for supportive therapy vary from one centre to the next. This international forum addresses these collection modalities. Eight centres responded to the survey. Granulocytes are collected from both, healthy donors and from patients’ relatives in five out of eight centres while in two centres only the patients’ relatives serve as granulocyte donors and 1 centre collects granulocytes exclusively from healthy volunteers. Cytomegaly (CMV)-negative donors are preferred for CMV-negative patients. In one centre the donors (whole blood donors) are not routinely tested for CMV. Mobilization regimens and donation frequencies differ among the centres, but G-CSF is commonly used in all centres. The dose varies between 5 lg ⁄ kg body weight (BW) and 12 lg ⁄ kg ⁄ BW 8–18 hours before donation. In two centres a standard dose of 300 lg is given. Although the combination of cytokines and steroids for donor stimulation is favoured by several authors [3], in only half of the centres this regimen is used. The main reason to refrain from the combination of cytokines and steroids was to minimize cumulative adverse events. In three centres G-CSF is used as the only stimulating agent. Prednisolone is used at a dose of 50 mg and dexamethasone at a dose of 3 or 8 mg, either in combination with G-CSF or alone. Of note, the administration of G-CSF is only allowed for the mobilization of peripheral stem cells in healthy donors in two out of eight centres. Thus, healthy volunteers are pretreated exclusively with steroids in one of these centres, while in the other one the short-term application of G-CSF for granulocyte collection requires an approval from the local ethics committee as well as a donor insurance. The frequency of granulocyte donation is restricted to a few times in all centres. It ranges from once in a lifetime to five times per year. In three centres the number of donations is restricted to three (two centres) or four donations (one centre) in a lifetime, respectively. In one centre the number of donations differs between healthy volunteers (once in a lifetime) and patients’ relatives (twice in a lifetime, if necessary both donations within 1 week]). In two centres the number of donations depends on the mobilization regimen used. In the 1st centre G-CSF primed donors are allowed to donate five times per year and steroid primed donors are allowed to donate only four times a year. These restrictions are in accordance with their national law allowing the exposure of donors to 150 g HES within a year and to 200 mg prednisolone per year. In the second centre, donors, pretreated with G-CSF alone, may donate four times per year, but only twice a year if treatment is combined with steroids. In only three of eight centres high molecular weight HES is still used whereas two centres use low molecular weight HES (130 kD) for granulocyte collection. In one centre there is no preference for either, and in two centres no sedimentation agents are used at all; of the latter, one only provides granulocytes for children. In two centres, in which there was a switch from high molecular weight to low molecular weight HES, the efficiency of collection became less resulting in significantly lower granulocyte yields, and the granulocyte collection modalities of the cell separator were therefore adjusted in one of these centres. In all but one centre ABO compatibility between donor and recipient is respected. In only one centre transfusions are given, even against the blood group barrier. In case of blood group incompatibility the product is either red cellor plasmadepleted or both. Another centre demands absence of haemolysins and an alloagglutinine-titre below 1:128 in case of minor incompatibility. In four centres the RhD status of the patients is respected irrespective of their gender and age. In two centres anti-D prophylaxis is given in case of RhD incompatible granulocyte Vox Sanguinis (2010) 98, 567–575

The development of AIDS or AIDS‐related conditions in a cohort of HIV antibody‐positive homosexual men during a 3‐year follow‐up period

Abstract. One hundred and thirty‐three homosexual men seropositive for the antibody against human immunodeficiency virus (HIV) were enrolled in a prospective study in 1984–85. The 3‐year cumulative incidences of the acquired immunodeficiency syndrome (AIDS) and AIDS‐related conditions, by life‐table analyses, were 18% and 34%. The cumulative incidence of immune deficiency defined as CD4 lymphocytes < 0.5 times 109 l−1 was 70% at 3 years. Absence of antibodies to p24 antigen, HIV antigenaemia, CD4 lymphocytes < 0.3 times 10 l−1 and elevated serum level of IgA were significantly associated with the development of AIDS. There was no association between disease progression and persistent generalized lymphadenopathy.

Limiting dilution analysis of interleukin-2 producing helper T-cell frequencies as a tool in allogeneic hematopoietic cell transplantation.

Background. A reliable in vitro test that estimates the level of ongoing alloreactivity would be valuable in allogeneic hematopoietic cell transplantation (HCT) as a help to guide clinical interventions such as donor lymphocyte infusions and changes in the immunosuppression. In the present study, the use of limiting dilution analysis of interleukin-2 (IL-2) producing helper T lymphocyte frequencies (HTL assay) as a way to quantify alloreactivity following HCT was investigated. Methods. Serial HTL assays were performed following allogeneic HCT with myeloablative or nonmyeloablative conditioning in 26 patients with hematologic malignancies. Results. Deviations from single-hit kinetics were frequently observed in the HTL assays and a nonlinear model was therefore used for analysis. The results of this analysis suggested the presence of an inhibitory cell population. Inhibition was observed in the majority of patients and was not restricted to a specific transplant regimen. Inhibition occurred more often with high frequencies of IL-2 producing cells, indicating a physiological role of the putative inhibitory cell population in the regulation of an immune response. Higher frequencies of IL-2 producing cells were observed in patients with acute graft-versus-host disease grades II-IV than in patients with grades 0-I (P = 0.046), indicating that the degree of ongoing alloreactivity is indeed quantified by the HTL assay. Conclusions. We find that the HTL assay may yield interesting insight into regulation of immune responses following allogeneic HCT, but because of the complexity of the results obtained, its use as a routine procedure to guide immunosuppression cannot be recommended.