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Purpose: From May 2003 to March 2005, 37 patients with haematological malignancies were transplanted with peripheral blood stem cells after nonmyeloablative conditioning with fludarabine 30 mg/m2 i.v. once daily on day −4 to −2 and 200 cGY of total body irradiation on day 0. Post-transplant immunosuppression consisted of oral cyclosporin 12.5 mg/kg/day and mycophenolate mofetil 30 mg/kg/day. Three patients were not eligible for follow-up due to lack of informed consent or informative markers for chimerism measurement. The purpose of this study was to evaluate kinetics of early chimerism of CD4+, CD8+, and, CD15+ cells immediately post transplant and furthermore to identify factors associated with rejection of graft. Methods: Blood samples were collected post transplant on day +1–3, +5–7, and then weekly. Samples were separated with immunomagnetic beads and DNA was salt extracted. Chimerism analysis was performed by automated high-resolution real-time quantitative PCR based on short insertion or deletion polymorphisms or single nucleotide polymorphisms. Results: The kinetics of CD15+ chimerism differed from that of CD4+ and CD8+ chimerism (Figure). CD15+ donor chimerism was low with a median of 0.9% donor cells on day +1–3, and remained low until around day +14. Subsequently the donor chimerism increased rapidly towards 100%. The CD4+ and CD8+ donor chimerism in contrast started higher with a median of 16.3% and 15.1% respectively on day +1–3, increased slowly, and took months to reach 100%. CD4+, CD8+ or CD15+ donor chimerism on day +1–3 did not predict later development of acute graft versus host disease. Two patients had primary rejection of the donor stem cells, while four patients had secondary rejection. The patients with primary rejection had no measurable donor chimerism in any cell lineages from day +1–3. No patient with CD15+ donor chimerism above the median (0.9%) on day +1–3 rejected, while 40% (6/15) of the patients with CD15+ donor chimerism below the median rejected (LogRank test, p=0.01). No differences were found in total number of nucleated cells, CD34+, CD3+, CD4+, or CD8+ cells in the donor harvest between patient who rejected and those who did not. Conclusions: The kinetics of early CD15+ donor chimerism differed from the kinetics of early CD4+ and CD8+ donor chimerism. Early CD15+ donor chimerism was markedly lower than CD4+ and CD8+ donor chimerism. CD15+ donor chimerism increased toward 100% donor chimerism more rapidly than the CD4+ and CD8+ chimerism. Primary rejection of donor stem cells can be identified early, as these patients have no measurable donor CD4+ or CD8+ chimerism on day +1–3. Furthermore, patients with CD15+ donor chimerism above the median on day +1–3 have a low risk for rejection. Figure Figure

Cytokines are thought to play an important role in the pathophysiology of Graft-versus-Host disease (GVHD). To study the relationship between cytokines and GVHD, we obtained peripheral blood mononuclear cells (MNC) from 21 patients with hematologic malignancies and their human leukocyte antigen identical sibling donors before and sequentially after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning. The MNC were cultured for 72 hours either alone or in mixed lymphocyte cultures with irradiated MNC of recipient, donor or HLA-mismatched third-party origin. The gene expression of interleukin (IL)-2, IL-4, IL-10, IL-18, tumor necrosis factor-α and transforming growth factor-β in each culture was then measured by real-time quantitative RT-PCR. The composition of the responder MNC differed between patients and donors and changed following HCT with a possible influence on the results. Early after transplant (day +14) the IL-10 mRNA level in response to recipient or donor antigens was higher in patients who did not develop clinical significant acute GVHD when compared to the level in patients who subsequently developed acute GVHD grades II–IV (p=0.005 and p=0.004, respectively). The IL-10 mRNA level on day +14 was highly correlated to the pre-transplant mRNA level of the recipient MNC but not to the level of the donor MNC, suggesting that the IL-10 mRNA detected on day +14 originated from responder cells of recipient origin. A higher IL-10 mRNA level was found in MNC obtained pre-transplant from recipients who progressed or relapsed after the transplant when compared to the level in patients who did not, p=0.03. In conclusion we find that detection of cytokine gene expression in MNC by real-time quantitative RT-PCR is an interesting tool in the study of the immune responses following HCT. A number of factors are likely to influence the results obtained and in this study we have identified the composition of the MNC as such a factor. Our results suggest a role for IL-10 as an inhibitor of alloreactivity following nonmyeloablative HCT. This inhibition may have dual effects by limiting the degree of acute GVHD and at the same time increase the probability of relapse.

We have studied whether the decreased lymphocyte proliferative responses of AIDS lymphocytes to stimulation by mitogens and antigens may be overcome when challenged with a combination of calcium ionophore A23187 and phorbol ester PMA. Comparison of the proliferative response of lymphocytes from nine patients with AIDS with the response of lymphocytes from nine control subjects showed that the response of AIDS lymphocytes was severely decreased when stimulated with PHA and no further response could be achieved by stimulation with A23187/PMA. On the other hand, no significant difference between the PHA-induced rise of cytoplasmic free calcium concentration ([Ca2+]1) in normal and AIDS lymphocytes was observed. The percentage of cells expressing IL-2 receptors (CD25) was also normal both after addition of PHA and after addition of A23187/PMA and the expression was normal on both CD4 and CD8 cells. The production of IL-2 in normal lymphocytes stimulated with A23187/PMA was 33 times higher than that after stimulation with PHA. In AIDS lymphocytes the production of IL-2 induced by all activators was severely decreased compared to control subjects, although the production of IL-2 after stimulation with A23187/PMA was higher than that in control lymphocytes after stimulation with PHA. The present study shows that a direct activation of protein kinase C combined with mobilization of cytoplasmic calcium does not overcome the lymphocyte proliferative deficiency of AIDS lymphocytes.

This study was undertaken to clarify the mechanism behind the severely decreased lymphocyte proliferative response upon stimulation with mitogens and antigens seen after allogeneic bone marrow transplantation (BMT) in man. We investigated eight BMT patients and eight controls and found that the proliferative response of patient cells was reduced both when the cells were stimulated with phytohaemagglutinin (PHA) and when they were stimulated with a combination of phorbol myristate acetate (PMA), which is an activator of protein kinase C (PKC), and the calcium ionophore A23187, which irreversibly opens for calcium transport into the cell (median relative responses were 41 and 37%, respectively). However, the PHA-induced increase in the concentration of intracellular free calcium in post-BMT cells was not significantly different from the values found in control cells and the expression of interleukin 2 (IL-2) receptors (CD25) was only slightly decreased. However, the production of IL-2 was severely decreased in patient cells after stimulation with A23187/PMA (median 3541 units), although it was higher than in PHA-stimulated control cells (median 354 units). These results show that a direct activation of PKC by PMA combined with an increase in intracellular free calcium by A23187 cannot overcome the lymphocyte proliferation deficiency in cells from patients after allogeneic BMT. The data suggests that the defect is affecting the diacylglycerol pathway considerably more than the inositol triphosphate pathway.

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.

The aim of the present study was to investigate the relation between the histopathological findings in LAV/HTLV-III lymphadenitis and immunological, clinical, and serological variables. The study group included 38 consecutive homosexual men with persistent generalized lymphadenopathy (PGL) in whom lymph node biopsy was performed. The histopathological lymph node changes were grouped into three stages. Opportunistic infections at the time of biopsy and their development during follow-up were significantly associated with stage III histology (follicular depletion). Analysis of blood from 10 patients with stage III histology revealed significantly (P less than 0.01) decreased proliferative responses of lymphocytes to mitogens and reduced absolute number of CD5+ and CD4+ lymphocytes compared with 17 patients with stage I histology (follicular hyperplasia), whereas patients with stage II histology (follicular involution) had intermediate values. The absolute number of CD8+ lymphocytes was increased in all three stages, as was IgG, while increase in IgM and IgA was restricted to stage III. No difference was observed between the different histopathological stages with respect to the specificity of the anti-LAV/HTLV-III antibody as measured by immunoblotting. In conclusion, the defects of lymphocytes from the blood of LAV/HTLV-III infected persons reflect alterations in secondary lymphoid tissue. Further, there is a close correlation between these alterations and the clinical status of the patients.

Retroviral infections are accompanied by immunosuppression in a variety of species. For feline leukemia virus, the immunosuppression has been ascribed to the transmembrane envelope protein, p15E, which suppresses the proliferative responses of cat, mouse, and human lymphocytes. A similar suppressive effect has been shown for a lysate of human immunodeficiency virus (HIV), strain HTLV-IIIB. Here we determined that detergent-disrupted HTLV-IIIB lysate exerted a strong suppressive effect on PHA-stimulated lymphocytes. Preparations of whole virions, a lysate of a local HIV isolate grown on MP-6 cells, and a commercially obtained UV and psoralene-inactivated lysate were examined and demonstrated to have a similar suppressive effect. The HIV lysate was not directly cytotoxic to lymphocytes and did not contain tumor necrosis factor or lymphotoxin. The HIV lysate specifically suppressed the proliferation of a range of hemopoietic cell lines from man and mouse including three EBV transformed CD4and IL-2 receptor-negative B-cell lines. The lysate also suppressed the formation of human bone marrow colonies, whereas the lysate had only a slight or no effect on fibroblasts. The suppression of lymphocyte proliferation was not abrogated by addition of IL-2 or IL-1 and the HIV lysate inhibited the expression of IL-2 receptors on suboptimal PHA-stimulated mononuclear cells. The suppressive factor(s) has not been characterized in molecular terms, but suppressive activity was recovered in fractions with a molecular weight of about 67,000 and in both the glycoprotein fraction and in the glycoprotein-depleted fraction of the HIV lysate. Sera from one-third of a small series (N = 13) of individuals with antibodies to HIV seem to be able to neutralize the suppressive properties of HIV lysate in cultures.

The prognostic value of various immunologic tests was investigated in 150 HIV-seropositive homosexual men, who were initially without HIV-related symptoms or AIDS and who were followed for a median of 12 months (range 3-28 months). The laboratory investigations included HIV antigen in serum, total lymphocyte count, T-helper (CD4) and T-cytotoxic/suppressor (CD8) counts, and lymphocyte transformation responses to the mitogens phytohemagglutinin (PHA) and pokeweed mitogen (PWM), and to antigenic extracts from Candida albicans and cytomegalovirus. 24 individuals developed HIV-related symptoms or AIDS (11 cases). All parameters except the CD8 count were of prognostic value, but a multivariate analysis of symptom-free survival showed that HIV antigenemia, a CD4 count less than 0.5 x 10(9)/l, and relative response to PWM below 25% of controls contained all the prognostic information. Individuals abnormal at entry for these 3 variables had a theoretical 36 times as high hazard of developing symptoms within the observation period as had individuals with normal parameters. There was no significant covariation between HIV antigenemia on the one hand and CD4 count and response to PWM on the other. Although, the latter 2 variables covaried, each of them provided independent information, and both were used to classify the degree of the immunodeficiency in 3 stages: Im-0 with normal values, Im-1 with one, and Im-2 with both tests abnormal. Individuals in stage Im-2 had a 10 times increased risk of developing symptoms. The immunologic staging correlated significantly with the clinical grouping (CDC criteria). This staging improved in only 1, but deteriorated in half of 36 individuals observed for at least 18 months. Thus, the staging is likely to prove useful when attempts to arrest the immunodeficiency of HIV-infected individuals has to be monitored.

Lymphocytes from 16 AIDS patients were tested in the cell-mediated lympholysis assay (CML). The ability to produce alloreactive cytotoxic lymphocytes in vitro was found to be substantially reduced when compared with concomitantly investigated normal controls. Addition of interleukin 2 (IL-2) to the inducer cultures increased the cytotoxic activity, but not to normal levels. The CML response did not correlate with the relative or absolute number of Leu 3+ cells or the proliferation in effector suspensions. The ability to produce cytotoxic cells in CML, and the degree of potentiation by IL-2, was positively correlated with the absolute number of Leu 2+ cells in peripheral blood of the patients, which was below normal in 56% of the patients. It is suggested that the low CML in AIDS patients is primarily caused by defective T-cell help. In addition patients with decreased absolute numbers of Leu 2+ cells may have a reduced number of CTL precursors. The natural killer (NK) activity of AIDS lymphocytes was reduced, but could be improved by incubation with IL-2 in vitro. The mononuclear cells from the patients showed a decreased ability to respond and to stimulate in the mixed lymphocyte culture. In one of the AIDS patients, the CML was found to induce autoreactivity in vitro.

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 less than 0.0005), cytomegalovirus isolation (P less than 0.01), low skin test reactivity (P less than 0.01) and episodes of fever within the 2 month period prior to investigation (P less than 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 less than or equal to 1.0 was significantly more frequent in homosexual men who both had HTLV-III Ab and excreted cytomegalovirus (P less than 0.01). The H/S ratio of HTLV-III negative homosexuals were significantly lower than that of the controls suggesting that a non-HTLV-III related immunosuppression occurs among homosexuals. Within 2 years after the investigation AIDS or the AIDS related complex developed in three of the men, who at the first investigation all had HTLV-III Ab, alterations in T-lymphocyte subsets and cutaneous anergy. It is suggested that a combination of T-cell subset determination and determination of HTLV-III Ab may provide more valuable prognostic information than isolated determination of HTLV-III Ab.

OBJECTIVE--To investigate the impact of the clinical course of the primary HIV infection on the subsequent course of the infection. DESIGN--Prospective documenting of seroconversion, follow up at six month intervals, and analysis of disease progression by life tables. PATIENTS--86 Men in whom seroconversion occurred within 12 months. PRIMARY OUTCOME MEASURE--Progression of HIV infection, defined as CD4 lymphocyte count less than 0.5 X 10(9)/l, recurrence of HIV antigenaemia, or progression to Centers for Disease Control group IV. MAIN RESULTS--Median follow up was 670 (range 45-1506) days. An acute illness like glandular fever occurred in 46 (53%) subjects. Three year progression rates to Centers for Disease Control group IV was 78% at three years for those who had longlasting illnesses (duration greater than or equal to 14 days) during seroconversion as compared with 10% for those who were free of symptoms or had mild illness. All six patients who developed AIDS had had longlasting primary illnesses. Three year progression rates to a CD4 lymphocyte count less than 0.5 X 10(9)/l and to recurrence of HIV antigenaemia were significantly higher for those who had longlasting primary illnesses than those who had no symptoms or mild illness (75% v 42% and 55% v 14%, respectively). CONCLUSION--The course of primary infection may determine the subsequent course of the infection.

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 manufacturer's instructions, using a low cut-off value defined as (4N + P)/5, where N and P 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.

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 less than 0.5 x 10(9) l-1 was 70% at 3 years. Absence of antibodies to p24 antigen, HIV antigenaemia, CD4 lymphocytes less than 0.3 x 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. When adjusted to the probable year of infection, these results are in accordance with previous cohort studies. It is concluded that most, or all, subjects seropositive for HIV will develop progressive loss of CD4 lymphocytes followed by clinical signs of immune deficiency, and that differences among previous cohorts with respect to disease progression are probably due to differences in the duration of infection.

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.

Screening of 70 asymptomatic homosexual men in Copenhagen revealed that 13 (19%) had T-helper to T-suppressor (H/S) ratios less than or equal to 1.0. Clinical and immunological follow-up examinations for 2-7 months (mean, 5.2 months) disclosed that none of the 13 men developed the acquired immunodeficiency syndrome (AIDS) or AIDS-like symptoms. An increase in H/S ratios to greater than 1.0 was observed in 11 of the 13 men during the time of observation. The decreased H/S ratios were due to an increase in the T-suppressor population. The T-helper population did not at any time differ from that found in 31 male control subjects. The biological relevance of the observed decrease in H/S ratios was supported by the demonstration of a positive correlation to a decrease in the proliferative response of the lymphocytes. Serological studies did not reveal any specific infectious background for the low H/S ratio found in the 13 men, and the reason for the 'spontaneous' increase during the time of observation remains unknown. The present results indicate that most asymptomatic homosexual men with a decreased H/S ratio will experience normalization of the immunological variables.

T-cell and B-cell functions were studied in 35 patients with untreated multiple myeloma (MM) and in 16 patients with MM treated with prednisolone, melphalan and vincristine. The numbers of CD4+ T cells were normal in untreated MM patients, but markedly decreased in treated patients, whereas CD8+ cell numbers were normal in untreated and treated patients. Mitogen-induced as well as antigen-induced lymphocyte proliferative responses were reduced, but not further affected by treatment. The antigen-induced proliferative responses by lymphocytes of treated, but not of untreated patients, correlated positively to the proportions of CD4+ cells among MNC. Taken together, the findings suggest selective loss of CD4+ subpopulations during cytotoxic treatment. Pokeweed mitogen (PWM)-induced Ig production was generally low, but significantly reduced Ig production was only seen in experiments employing MM B cells and monocytes co-cultured with irradiated T-enriched cells. Irradiated MM T cells displayed normal helper function when co-cultured with normal B cells stimulated with PWM. MM B cells and monocytes cultured with irradiated normal T cells produced little Ig; however, MM monocytes were not suppressive. In 2 of 3 patients with either IgG-kappa or IgA-kappa myeloma, the numbers of PWM-stimulated B cells that produced kappa chains were somewhat higher than those found among normal MNC. The impaired ability of antibody production by B cells from untreated MM patients seems to relate to intrinsic B cell defect(s) rather than to abnormal regulation by T cells or monocytes. However, disturbances in the functions of CD4+ cells may be observed in treated MM.

Screening of sixty-six asymptomatic homosexual men from Copenhagen revealed significantly lower Leu-3a/Leu-2a ratios as compared to controls. Ten (15%) of the homosexuals had a ratio less than or equal to 1.0. The low Leu-3a/Leu-2a ratios were the result of an increase in the absolute number of Leu-2a cells. Homosexuals with many partners and those who had been sexual partners of patients with the acquired immuno-deficiency syndrome (AIDS) had significantly lower ratios than those without these features. Cytomegalovirus was isolated from urine and/or sputum of 15% and this was associated with a Leu-3a/Leu-2a ratio less than or equal to 1.0. The observed immunological abnormalities could either represent latent infection with the putative AIDS agent or alternatively be caused by repeated infections and/or exposure to allogenic spermatocytes or lymphocytes.

The titers of IgG and IgA to Pneumocystis carinii in 36 AIDS patients did not differ significantly from those in 31 controls. Only 2/15 patients (13%) with P. carinii pneumonia (PCP) had titers of IgM antibodies greater than or equal to 5, which is significantly less frequent than in 32 controls (62%) and in 21 AIDS patients without PCP (43%). The risk of PCP was 5 times higher in patients without IgM antibodies to P. carinii than in patients who had these antibodies. A significantly higher percentage of those without PCP (57%) showed increasing titers of IgM antibodies to P. carinii in the second of paired samples taken about 6 months apart, compared with whose with PCP (9%; p = 0.05). All patients had high titers of antibodies to CMV and HSV and normal total concentrations of immunoglobulins. None of the patients responded in lymphocyte transformation to P. carinii, CMV, or HSV antigens. There is no obvious explanation to the selective lack of IgM antibodies to P. carinii in patients with PCP. Lack of IgM antibodies may be a marker for an immunodeficiency to P. carinii.

An individual, BK, repeatedly gave typing responses against homozygous typing cells representing more than two HLA--D antigens. Family studies showed that she had inherited the HLA--Dw2 and Dw7 determinants, in agreement with results from primed lymphocyte typing. The HLA--A, B, C types of the stimulators giving rise to unexpected typing responses all involved the HLA--A1, A3 or A11 antigens. The hypothesis of this specificity in low-responsiveness was confirmed in the independent stimulator sample provided by the 7th workshop homozygous typing cell panel. The same pattern was observed when BK was tested as a responder towards related and unrelated heterozygous stimulators. Furthermore, in three-cell experiments it was found that BK cells were able to suppress the response to the Dw-identical individual, BS, towards stimulators carrying HLA--A1, A3, or A11. This effect of BK's cells appeared to be radiosensitive. We were not able to demonstrate suppressive supernatant factors in the relevant cultures, neither were we able to find circulating cytotoxic cells by the direct CML-technique, nor an accelerated cytotoxic response by the indirect CML-technique against targets carrying HLA--A1 or A3. This is the first demonstration of induction of suppressor cells in MLC by HLA--A, B, C antigens in the stimulator cells.

Abstract 2397 Introduction. DMSO is the universally used cryoprotectant for freezing peripheral blood stem cell for autologous transplantation. DMSO has a significant side effect profile including vasoconstriction, nausea and vomiting and with transplant related mortality now in the region of 1–3% avoiding DMSO toxicity is of increasing importance. Methods. The study was questionnaire based looking at centres freezing strategy(s), administration of stem cells to patients and individual forms for each patient transplanted with follow up form for side effect analysis. Side effects were defined using NCI criteria (version 3.0) and assigned to DMSO (or not) by the reporting centres. On account of substantial centre variation noted at initial analysis results were analysed using the Mantel-Haenszel Odds Ratio (OR) averaged over the centres. Results. 64 centres contributed data for a maximum of 12 months between January 2007 and December 2008. A total of 1651 patients, 1008 male and 637 female, age range 18–76, median 56, with 875 myeloma patients, 540 NHL, 220 Hodgkin‘s Disease and 16 CLL were used for analysis. Almost all myeloma and most of the other patients had melphalan based regimens. From the Centre survey it was noted that 51 centres used 10% DMSO (1273 patients) but on account of multiple centre strategies 15 centres used concentrations down to 5%. Three Centres using 10% DMSO washed cells before infusion while another 9 Centres washed cells occasionally (>10% of transplants). The median amount of DMSO given per patient was 20ml although the upper limit per Centre was often very much higher. 75% of Centres did not use any delay between bags of stem cells and the median duration of infusion was 22 minutes. 440 patients experienced nausea or vomiting related to DMSO, 123 hypo- or hypertension and 84 other side effects. The side effect profile was then amalgamated so that no patient was counted more than once. DMSO was calculated in ml/Kg body weight for each patient then split into quartiles for analysis. The OR for any side effect assigned to DMSO between patients in the highest quartile and patients in the lower three quartiles averaged over age and disease classification was 1.7 (Confidence Interval 1.2 to 2.4, p = 0.003). When split into groups by disease (lymphoma or myeloma) and age (>median and Conclusions. DMSO contributes to the morbidity of patients undergoing autologous transplantation. Strategies to reduce DMSO exposure can reduce these complications. Disclosures: No relevant conflicts of interest to declare.

Flow cytometric analysis of the peripheral blood mononuclear cells in a six year old girl with a primary cellular immune deficiency showed a normal fraction of CD3 positive T cells. Most (70%) of the CD3 positive cells, however, expressed the gamma delta and not the alpha beta T cell receptor. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that most of the gamma delta T cell receptors existed as disulphide-linked heterodimers. Proliferative responses to mitogens were severely reduced, but specific antibody responses after vaccination could be detected. A thymic biopsy specimen showed severe abnormalities of both the thymic lymphoid and epithelial component with abortive medullary differentiation and almost an entire lack of Hassall's corpuscles. This patient represents a case of primary immune deficiency syndrome not previously described. Thymic deficiency associated with a high proportion of T cells expressing the gamma delta T cell receptor has been described in nude mice, and it is suggested that the immune deficiency of this patient may represent a human analogue.

HIV antigenemia was found in 52/243 HIV antibody positive individuals attending 2 AIDS-screening clinics, giving a prevalence of 13, 25 and 76% in CDC groups II, III and IV, respectively. No correlation was found to decreased CD4 lymphocyte values in the individual groups. HIV antigen therefore identified a separate subpopulation. For 138 asymptomatic patients followed prospectively both laboratory parameters predicted HIV-related events, the relative risk factor being 4 for low CD4 value and 6 for presence of HIV antigen. Individuals presenting with HIV antigen and decreased CD4 count all developed disease within 18 months, the relative risk factor being 24. Thus the 2 markers, when measured together, effectively separated asymptomatic HIV-infected patients into 1 of 3 risk categories.

An effective method for specific depletion of mature T lymphocytes from human bone marrow mononuclear cells (BMMC) with preservation of prethymic T cells and natural killer (NK) cells is presented. The BMMC were incubated with F101.01, a monoclonal antibody recognizing an epitope of the T-cell receptor-CD3 complex, and subsequently with immunomagnetic beads. Flow cytometric analysis demonstrated that mature T cells were efficiently depleted and that NK cells and prethymic T cells were preserved in the BMMC. Furthermore, T cell-mediated immune reaction as measured by thymidine incorporation after stimulation with phytohaemagglutinin was abolished, whereas NK activity as measured by 51Cr release using K562 as target cells was preserved. Recovery of colony-forming units of granulocyte-macrophages was 60-70%.

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 × 10 8 [range (6.8-29.6) × 10 8 , 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 × 10 8 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 × 10 8 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.

Recently, much interest has focused on the role of HLA class II antigens in T cell-T cell interactions. We have studied the stimulatory capability in the primary mixed leukocyte reaction and the primed lymphocyte reaction of 11 alloactivated, HLA-DR- or -DP-reactive CD4-positive T-cell lines (Ta). From 70 to 90% of the Ta were HLA class II-positive as judged by the reactions with HLA class II-reactive monoclonal antibodies, and the Ta carried the DR allospecificities of the original T-cell donor when typed in the microcytotoxic test using DR-specific alloantisera. Neither irradiated nor nonirradiated Ta stimulated primed lymphocytes directed against the relevant HLA class II antigens on the Ta. Interferon-gamma, recombinant interleukin 1, phorbol myristate acetate, calcium ionophore, and adherent cells had no effect on the stimulatory capability of Ta. The ability of irradiated Ta to stimulate in the primary mixed leukocyte reaction (median counts per minute (cpm) 5.5 x 10(3] was significantly lower than that of peripheral blood mononuclear cells (cpm: 44.0 x 10(3]. The stimulation by Ta was almost only seen when the Ta were specifically directed against the class II antigens of the responder peripheral blood mononuclear cells (i.e., in combinations with "backstimulation") (median cpm: 21,000). In mixed leukocyte reaction combinations without backstimulation, significantly weaker reactions were seen (median cpm: 1,000). This observation may explain previous controversies concerning the stimulatory capacity of Ta. Recombinant interleukin 2 significantly enhanced the very low mixed leukocyte culture responses induced by class II-incompatible Ta in combinations without backstimulation but had no significant effect on cultures with Ta autologous to the responder peripheral blood mononuclear cells. Thus, allogeneic class II-positive Ta can induce interleukin 2 responsiveness but lack accessory cell function(s) necessary for the induction of interleukin 2 production in primed and unprimed T cells.