Disturbances in iron utilization in acute leukemia and preleukemia]

With Prussian blue reaction nonhaemoglobin iron in the erythroblasts is demonstrable. Three pathological sideroblast types are recorded separately: abnormal intermediate type I and II sideroblasts and ring sideroblasts, representing increasing levels of sideroachrestic disturbance. This permits the classification of sideroachrestic disturbances into four degrees of seriousness. The frequency of a sideroachrestic disturbance in 47 untreated patients with acute myeloid leukaemia was 87%. Among 11 patients with preleukaemic condition, 8 had a disturbance of iron utilisation. In both preleukaemia and leukaemia mainly intermediate sideroblasts were present. All patients with preleukaemia developed leukaemia within 1-20 months. In the course of preleukaemic condition a slight increase of iron misutilisation was obvious when terminating in overt leukaemia. This could be of prognostic importance. After treatment, pathological sideroblasts disappeared only in 2 out of 15 patients with complete remission. There was no correlation between effect of therapy and course of iron misutilisation.     

Plasma transcobalamins in haematological disorders

Plasma UBBC-B12 and transcobalamins were measured in 112 patients suffering from different haematological disorders. The data showed different patterns of changes in plasma transcobalamin profile in different haematological disorders. Plasma UBBC-B12 and transcobalamins were significantly higher than normal in untreated chronic myeloid leukaemia, acute promyelocytic leukaemia, nutritional megaloblastic anaemia and in refractory anaemias with hypercellular marrow. Normal levels of these proteins were noted in chronic lymphatic leukaemias, in primary and secondary hypereosinophilic states and in multiple myeloma. Subnormal levels of these proteins were observed in hypoplastic anaemia and acute lymphoblastic leukaemia. Chronic myeloid leukaemia patients during blast crisis and acute myeloid leukaemia patients except those suffering from acute promyelocytic leukaemia showed varying pattern of plasma transcobalamins depending on type of blast crisis or FAB subtype of AML. The significance of these changes in plasma transcobalamins have been discussed along with the experience of other workers in this field.     

Changes in surface antigens of HL-60 cells during differentiation in vitro

We have examined the pattern of binding of monoclonal antibodies OKM 1, FMC 10, FMC 12, FMC 13, FMC 17 and FMC 33 to human promyelocytic leukaemia (HL-60) cells. We found that the expression of antigens detectable with FMC 17 and FMC 33 (specific for monocytes and macrophages) was increased by exposure of HL-60 cells to 1,25-dihydroxyvitamin D3 but not by exposure of HL-60 cells to 12-tetradecanoyl phorbol-13-acetate (TPA). The antigen detected with the OKM 1 antibody was highly induced by TPA. The expression of granulocyte-specific antigens detected by FMC 10 and FMC 13 was increased during induction of granulocytic maturation; these antigens were retained during monocyte-macrophage differentiation of HL-60 cells. We conclude that in some cases the expression of particular antigens during maturation of malignant cells proceeds normally while in other cases antigenic differences between leukaemic and normal cells at equivalent levels of maturation can be detected.     

Effect of AML serum on normal human myeloid differentiation and hexamethylene bisacetamide-induced granulocytic differentiation of the human HL-60 promyelocytic leukaemic cell line.

The effect of serum from 32 AML patients on the normal human myeloid differentiation and the hexamethylene-bisacetamide induced granulocytic differentiation of HL-60 promyelocytic leukaemic cell line was studied. Nonadherent normal mononuclear marrow cells were cultured in vitro at a concentration of 5 x 10(5) cells/ml for 6 days with each of the 32 AML sera. Ten normal human AB sera were used as control. The results showed an inhibitory activity on both morphological and functional differentiation of normal human myeloid immature marrow cells by 29 out of the 32 AML sera tested. These 29 AML sera were added to cultures of HL-60 (2.5 x 10(5)/ml) leukaemia cell line which incorporated 2 mM hexamethylene-bisacetamide for 6 days. The results showed no significant inhibition of hexamethylene-bisacetamide induced granulocytic differentiation by any of the 29 AML sera. The efficacy of hexamethylene-bisacetamide in inducing differentiation in the presence of inhibitory factors suggests a possible role in the treatment of AML patients.     

Cytogenetic analysis and clinical significance of chromosome 7 aberrations in acute leukaemia

The analysis was performed on bone marrow cells derived from 96 patients with acute leukaemia (AL): 76 with acute myelogenous leukaemia (AML) and 20 with acute lymphoblastic leukaemia (ALL). Aberrations of chromosome 7 were revealed in 20 (21%) of 96 analysed cases: in 14 (18%) with AML and in six (30%) with ALL. Structural aberrations, present in 13 patients (eight with AML and five with ALL), were unbalanced and led to partial monosomy (12 cases) or trisomy (four cases) of chromosome 7. Twelve (86%) out of 14 AML and all the ALL patients with chromosome 7 aberrations had complex karyotypes in their bone marrow cells. Monosomy 7 and 7q losses were frequently observed in the AML group, whereas, in the ALL group, gains in 7q and losses in the short arms constituted most chromosome 7 aberrations. The occurrence of monosomy, or of losses in 7q, results in a worse response to induction therapy in AML patients. The complete remission (CR) rate was significantly lower in this group in comparison to the group of AML patients with a normal karyotype (p = 0.01) in bone marrow cells.     

Generation of cytotoxic T lymphocytes from peripheral blood of leukaemic patients

Peripheral blood mononuclear cells from 13 patients with acute leukaemia were used to establish long-term interleukin-2-dependent cytotoxic T lymphocytes. Cells were grown in RPMI medium containing interleukin-2 (IL-2, 100 U/ml) and 2.5% conditioned medium prepared by activating normal lymphocytes with phytohaemagglutinin. Proliferation of IL-2-dependent CD3-positive lymphocytes was seen in 1 of 2 acute lymphocytic leukaemia cases (ALL), 1 of 4 acute myelogeneous leukaemia cases (AML) (M1) and 8 of 8 more differentiated AML. In 2 cases with detectable leukaemic cell markers (1 ALL and 1 AML) passageable cells were developed, that expressed normal T cell phenotypes (namely CD3, CD4, and CD8) at the expense of leukaemic cells. In 1 of 2 cases, long-term IL-2-cultured cells showed specific cytotoxic activity against autologous leukemic cells. The percentage killing against autologous and two allogeneic target cell lines at a 50/1 effector/target (E/T) ratio was 42%, 9% and 19% respectively. Similarly the cytotoxic activity of IL-2 activated from 4 different individuals against conventional tumour targets K562 and Daudi at a ratio of 50/1 was 29%–68% (median=55%) and 34%–78% (median=61%) respectively. It was also found that this killing potential of the activated cells was maintained for as long as culture was continued (median 23 days, range 17–75 days). The mechanism(s) of T cell proliferation at the expense of leukaemic blast cells in the case of a minority of leukaemic patients and the possible clinical therapeutic potential of these cells following in vitro IL-2 activation deserve further investigation.     

Immunoreactive calcitonin in leukaemia.

A radioimmunoassay was used to measure concentrations of immunoreactive human calcitonin (HCT) in plasma and leucocytes from patients with various leukaemic and myeloproliferative disorders. Plasma immunoreactive HCT concentrations were increased in 32 out of 33 patients with chronic granulocytic leukaemia (CGL) and in all eight patients with acute myeloid leukamia (AML) at presentation or in relapse. Out of 11 patients with other myeloproliferative disorders, eight had increased plasma immunoreactive HCT concentrations. Buffy-coat-cell extracts and culture media from peripheral leucocytes of patients with CGL also contained increased immunoreactive HCT concentrations. In contrast, plasma from patients with chronic lymphocytic leukaemia, acute lymphoblastic leukaemia, and AML in remission had low or undetectable immunoreactive HCT concentrations. Increased plasma and cellular concentrations of immunoreactive HCT may be a consequence of abnormal proliferation of myeloid cells and might prove to be valuable in predicting relapse in patients with myeloid leukaemias.     

TdT expression in acute myeloid leukaemia. Haemopoietic immaturity or maturational asynchrony?

A total of 412 cases of acute leukaemia were examined for the presence of nuclear terminal deoxynucleotidyl transferase (TdT) by indirect immunofluorescence. Of the 129 cases of acute myeloblastic leukaemia (AML FAB groups M1/M2) examined, 18% (n = 23) had significant proportions (greater than 10%) of TdT-positive blasts. Although most of these AML cases (n = 18) were of poorly differentiated (M1) type; 5 cases of AML showing features of granulocytic differentiation (M2) were also found to be TdT-positive. Even though TdT was generally more strongly expressed in the M1 group and associated with other markers of myeloid immaturity (Ia positive and lack of chloroacetate esterase), there was no inverse relationship with Sudan black or myeloperoxidase activity. In addition, although the proportion of AML-M1 cases with increased TdT-positive cells was slightly higher (18/95, 19%) than for the AML-M2 group (5/34, 15%) the results suggest that the presence of nuclear TdT in leukaemic myeloblasts may not only reflect cellular immaturity but may also be due to maturational asynchrony in otherwise well-differentiated blasts.     

Bacterial glutaminase in treatment of acute leukaemia.

A glutaminase-asparaginase enzyme from Achromobacter sp has antitumour activity in vitro and in animals. Glutaminase was administered in doses of 3500-20 000 IU/m2 body surface area/day to six patients with acute lymphoblastic leukaemia (ALL) and three patients with acute myeloid leukaemia (AML). The enzyme had a blood half life of 80 minutes but depletion of blood glutamine persisted for 12 hours after single doses. Seven patients, including four (two with AML and two with ALL) resistant to asparaginase, received repeated doses of glutaminase. Antileukaemic effects were observed in all seven; one elderly patient developed metabolic acidosis. Study of this new antileukaemic agent in patients with acute leukaemia at an earlier stage of their disease is now justified.     

Immunophenotyping of acute myeloid leukaemia: relevance of analysing different lineage-associated markers

The immunophenotype of 135 previously untreated patients with FAB defined acute myeloid leukaemia (AML) was studied at diagnosis. The panel of reagents included monoclonal antibodies (MoAb) recognising myeloid-associated determinants (CD11, CD13, CD14, CD33 and others) as well as MoAb directed towards lymphoid antigens (CD7, CD10, CD19) and TdT. The results indicate that CD13 and/or CD33 are consistently expressed in AML and only rarely in ALL blasts (131/135 + ve cases, versus 4/130 in ALL). Lymphoid antigen expression was rarely detected when CD10 and CD19 were investigated in AML (0.9% and 2% + ve cases, respectively), whereas significant positivities were found for TdT and CD7 (20% and 10% respectively). Concerning FAB subtypes, two new MoAb (LAM3 and LAM7) proved very useful in the specific recognition of AML with monocytic features. The phenotype CD13+ and/or CD33+, CD9+, HLA-DR- was found to be almost exclusive for M3 AML. The response to induction chemotherapy was analysed in CD7+ and in TdT+ patients. In the latter group a statistically significant lower response rate was found with respect to TdT-ve-AML patients.     

Familial Acute Myeloid Leukaemia with Acquired Pelger-Hu?t Anomaly and Aneuploidy of C Group

All five members of one generation in an Icelandic family were affected by acute myeloid leukaemia or preleukaemia. Two sibs died of acute myeloid leukaemia and another of myelofibrosis associated with leukaemic changes. The other two sibs are alive; one of them (the propositus) has haematological features consistent with preleukaemia; both of them have evidence of an abnormal cytogenetic clone in the bone marrow with 47 chromosomes, an extra chromosome in the C group. The finding of the acquired Pelger-Huët anomaly in the neutrophils of the five affected sibs suggests that a specific genetic cell defect has been transmitted with autosomal dominant characteristics. The presence of immunological deficiencies and an undue susceptibility to oncogenic viruses, as suggested by warts affecting three family members, may have played a part in the pathogenesis of the leukaemic process.     

Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity had no antiviral effects but moderately reduced lung inflammation

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT₅₀ value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable but low levels of antiviral antibodies after infusion. In comparison to the control animals, CCP-treated animals had similar levels of viral RNA in upper and lower respiratory tract secretions, similar detection of viral RNA in lung tissues by in situ hybridization, but lower amounts of infectious virus in the lungs. CCP-treated animals had a moderate, but statistically significant reduction in interstitial pneumonia, as measured by comprehensive lung histology. Thus overall, therapeutic benefits of CCP were marginal and inferior to results obtained earlier with monoclonal antibodies in this animal model. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.     

The pathomorphological diagnosis of acute leukaemias: cytology, cytochemistry and immunocytology

In 74 cases of acute leukaemia and of the blastic phase of chronic granulocytic leukaemia (CML), monoclonal antibodies of the "VI" series and a few commercially available antibodies proved to be of valuable help in establishing a definite diagnosis. In 4 out of 35 cases of AML (FAB-M 1-5), and in 2 out of 16 of the blastic phase of CML only the use of monoclonal antibodies secured the diagnosis. In the group of acute lymphoid leukaemias subtypes corresponding to various levels of differentiation, were defined. The blasts of 3 patients out of the 74 did not express any of the markers studied. Two additional cases were investigated for platelet peroxidase and studied with the antiplatelet antibodies VIPL 1, 2 and 3 by the electron microscope. These cases proved to be acute megakaryoblastic leukaemias (acute myelofibrosis).     

Bone Marrow Graft in Man after Conditioning by Antilymphocytic Serum

Allogeneic bone marrow grafts carried out after previous administration of antilymphocytic serum alone were attempted in 16 patients. Of these, six had acute myeloblastic leukaemia, four acute lymphoblastic leukaemia, and one a blast cell crisis in polycythaemia vera. Ten of these patients were in an overt phase of the disease and resistant to chemotherapy, while nine had complete agranulocytosis. In five of these patients erythrocyte and leucocyte antigenic markers demonstrated the establishment of the graft. One patient had thalassaemia major, and four others had aplasia of the bone marrow, in one case due to chloramphenicol poisoning and in another to virus hepatitis. The grafts were successful in the last two patients and transformed their clinical condition.No signs of early acute secondary disease were noted in any of the patients, either when the donor had been given antilymphocytic serum or when he was untreated. The grafts had no adoptive immunotherapeutic effect on the acute leukaemia. These observations have clearly shown that antilymphocytic serum has an immunosuppressive effect in man when it is used alone.     

Significance of detection of immune-complexed 8 kDa hydatid-specific antigen for immunodiagnosis of hydatidosis

Abstract Three micro-enzyme-linked immunosorbent assay (micro-ELISA) systems were developed and evaluated for detection of specific free circulating antigen and circulating immune-complexes (CICs) of 8 kDa antigen in the sera of patients with hydatidosis. All (100%) the sera of 30 confirmed positive cases of hydatidosis had detectable levels of antigen in the acid-treated sera. However, 23 (77%) and 26 (87%) sera of 30 confirmed cases had free as well as CICs of 8 kDa antigen in the untreated and in the polyethylene glycol (PEG) precipitated sera, respectively. None of the sera from other patients with parasitic infections or viral hepatitis had any detectable levels of 8 kDa antigen in the untreated, acid-treated or PEG-precipitated serum samples. The investigations, therefore, suggested that the demonstration of circulating antigen employing monospecific antibodies to affinity purified 8 kDa antigen in acid-treated sera is more efficient as compared to detection of free circulating antigen of CICs in the untreated or in the PEG-precipitated sera which could provide a specific immunodiagnostic tool for ongoing hydatid infection.     

IgA-antigliadin antibodies in IgA mesangial nephropathy (Berger's disease).

Circulating IgA-antigliadin antibodies were detected with enzyme linked immunosorbent assay (ELISA) in four of 121 patients (3%) who had IgA mesangial nephropathy and 14 of 17 children (82%) who had untreated coeliac disease. No positive cases were present in the 54 healthy subjects of the control group. Three patients who had IgA nephropathy and IgA-antigliadin antibodies underwent jejunal biopsy, and two showed mucosal atrophy. In these two patients urinary abnormalities, together with the IgA-antigliadin antibodies, disappeared completely after three months and five months, respectively, of following a gluten free diet. Circulating IgA immune complexes were found in most patients who had coeliac disease and Berger''s disease associated with IgA-antigliadin antibodies, suggesting overactivity of the B cells producing IgA in both conditions. By contrast, a circulating IgA rheumatoid factor was detectable in three of the four patients who had IgA nephropathy and asymptomatic coeliac disease but was always absent in children who had coeliac disease but did not show signs of renal disease. These results suggest that a more complex abnormality in the IgA immune response is necessary for renal disease to become manifest in patients who have gluten enteropathy.     

Studies on the presence of antibodies to EB virus and other herpesviruses in normal children and in infectious mononucleosis.

Children free from infectious disease have been examined by indirect immunofluorescence for the presence of antibodies to the intracellular capsid antigen of Epstein-Barr virus (EBV). In the first year of life 46%, between 2 and 6 years of age 66%, and between 7 and 14 years 91%, of the children proved positive. The corresponding percentages for the presence of antibodies to cytomegalovirus (CMV) and herpes simplex virus were about 50%, irrespective of the children's age. Serum samples from 69 patients suffering from infectious mononucleosis (IM) were tested for anti-EBV antibodies. Of the 29 Paul-Bunnell-positive patients 22 had antibodies, 11 of them in high titres (greater than 1 : 80). Of the 40 Paul-Bunnell-negative cases only 21 had antibodies, 8 in high titres. Of the Paul-Bunnell-negative cases, 73% were found to have anti-CMV antibodies, 32% in high titre. The respective percentages for the Paul-Bunnell-positive cases were 42% and 10%.     

Renal graft dysfunction during infection with cytomegalovirus: association with IgM lymphocytotoxins and HLA-DR3 and DR7.

Of 121 consecutive adult recipients of cadaver renal transplants who were treated with low dose steroids and azathioprine, 23 developed active cytomegalovirus infections. These 23 patients were divided into three groups on the basis of their symptoms related to the infection: five patients had no renal, respiratory, or haematological abnormalities; seven had renal dysfunction; and nine had renal dysfunction plus respiratory or haematological abnormalities. Two patients were regarded as a separate group because their infections occurred two to four weeks after graft nephrectomy. All but three of the patients produced IgM or IgG lymphocytotoxins during their infections. In the patients with mild infections and in control patients without infections, however, these lymphocytotoxins were predominantly IgG antibodies that were not precipitated by 3.5% macrogol (polyethylene glycol). In contrast, 12 of the 16 patients with renal dysfunction during their infections had broadly reactive IgM lymphocytotoxins. These IgM lymphocytotoxins lysed T as well as B lymphocytes at 22 degrees C and were precipitated by 3.5% macrogol, suggesting that they were circulating as immune complexes. Rheumatoid factors were found in sera from nine patients with cytomegalovirus infections, seven of whom developed leukopenia or pneumonia, or both, in addition to renal dysfunction. Some of these immune responses associated with cytomegalovirus infection in transplant recipients may be genetically controlled since 10 of 11 patients positive for HLA-DR3 or DR7 produced IgM lymphocytotoxins.