Monellin (MNEI) at 1.15 A resolution.

The X-ray crystal structure of a single-chain monellin protein (MNEI) has been determined at 1.15 A resolution. The model was refined to convergence employing anisotropic displacement parameters and riding H atoms to produce a final model with R(work) and R(free) values of 0.132 and 0.162, respectively. The crystal contains a single MNEI protein in the asymmetric unit and unusually lacks the dimer interface observed in all previous crystal structures of monellin and its single-chain derivatives. The high resolution allowed a more detailed view of MNEI than previously possible, with 38 of the 96 residues modelled with alternative side-chain conformations, including four core residues Thr12, Cys41, Leu62 and Ile75. Four stably bound negative ions were also located, providing new insight into potential electrostatic interactions of MNEI with the largely negatively charged surface of the sweet taste receptor T1R2-T1R3.

Monitoring myelomatosis.

There is evidence for the value of the following tests in the diagnosis, monitoring, and prognosis of myelomatosis: (1) serum paraprotein measurements, (2) urine paraprotein (including Bence Jones) measurements, (3) serum ablumin, (4) serum urea, (5) proteins in the urine other than those in 2, and (6) hemoglobin level. During treatment, increased rate of rise in 1 or 2, disproportionate increase in 2, emergence of related paraprotein, loss of 1 or 2 with reticulosarcomatous change, and monocytic leukemia suggest that more malignant subclones can emerge from the original myeloma clone, possibly due to drugs acting on DNA.

Standardisation of the mixed lymphocyte reaction.

The mixed lymphocyte reaction has been optimised with Ficoll--Triosil separated lymphocytes: 100,000 responding cells with 100,000 mitomycin-C-blocked stimulating cells, in 0.2 ml of TC 199 with 10% AB-negative known normal serum in Cook microplates. After incubation at 37 degree C for 120 h, 2 muCi of [3H]thymidine was added, harvesting after a further 15 h at 37 degree C. A pool of stimulating cells, freshly separated from 5 normal subjects (usable up to 36 h if kept at 4 degree C), produced a Gaussian normal range of increment in CPM from 17,000--88,000, with a between-batch coefficient of variation for triplicates of 22%. For tests against stimulating cells from a single donor a transformation index above 1.64 was significant.

Inhibition of thymidine uptake by staphylococci, a new method for the investigation of phagocytosis.

A radioassay employing (3H) thymidine, to measure inhibition of growth of Staphylococcus aures by human phagocytes is presented. The principle of this new method is that viable and dividing staphylococci take up thymidine more rapidly than white cells, so that whereas in control cultures containing staphylococci alone high counts per minute (cpm) are obtained within 90 min of incubation in test cultures both leucocytes and plasma from 25 normal subjects reduced the cpm, following ingestion and killing of staphylococci.

Combined detection of phenotype and Y chromosome by immunoenzyme labelling and in situ hybridisation on peripheral lymphocytes.

A novel staining method for simultaneously determining the immunophenotype and sex of peripheral lymphocytes is described. Cell surface markers on lymphocytes are identified using specific monoclonal antibodies located by a peroxidase anti-peroxidase staining technique (PAP). Lymphocytes are subsequently hybridised with a biotinylated Y chromosome-specific sequence probe and this is located by avidin-biotin-alkaline phosphatase staining. Following the two staining steps the preparation is examined and in the same field lymphocytes show brown peroxidase staining of cell surface markers and red staining of the Y chromosome. The application of this combined staining technique provides a convenient method for studying the development of different cell lineages following sex-mismatched bone-marrow transplantation and for the identification of chimeric situations. The method has been shown to be sensitive and reproducible.

Monitoring cytokine production in peripheral blood during acute graft-versus-host disease following allogeneic bone marrow transplantation.

Plasma concentrations and peripheral blood cells containing cytoplasmic cytokines were monitored during the post-transplant period in 10 patients who had received allogeneic bone marrow transplants (BMT) for the correction of inherited genetic disorders. The presence of CD14-positive cells containing cytoplasmic interleukin-1 alpha and beta in the peripheral blood was indicative of acute graft-versus-host disease (GVHD). Plasma concentrations of IL-1 alpha, IL-1 beta and TNF-alpha were significantly raised in the GVHD group when compared with the uneventful days. There was, however, poor temporal correlation between the plasma concentrations and clinical manifestations of acute GVHD. Cells containing cytoplasmic IL-6 were present in the peripheral blood when patients had clinically suspected and/or microbiologically confirmed infection. The results from this study demonstrate that analysis of peripheral blood cells for cytoplasmic IL-1 alpha and IL-1 beta are better markers of acute GVHD than is monitoring plasma concentrations of these cytokines.

Busulphan and cyclophosphamide cause little early toxicity during displacement bone marrow transplantation in fifty children.

Fifty children were induced with busulphan and cyclophosphamide before bone marrow transplantation. Our standard dose of cyclophosphamide was 2 g/m2 daily for 4 days, but this dosage was reduced if necessary so as not to exceed 75 mg/kg/day. A basal pulse rate that increased by more than 20 beats/min or a reduction in ECG voltage were indications for stopping cyclophosphamide. As a consequence, the fourth dose of cyclophosphamide was omitted for four children. One child died of cardiac arrest related to cyclophosphamide. We used busulphan at a dosage of 4 mg/kg/day for 4 days; neither 'busulphan lung' nor veno-occlusive disease occurred in any patient. The use of busulphan and cyclophosphamide did not guarantee freedom from interstitial pneumonitis, but this seemed to be related rather to the nature of prior treatment and the cytomegalovirus antibody status of the patient. Thus, we have developed a safe chemotherapy schedule for transplantation that avoids the need for radiotherapy and can, if necessary, be repeated after a 3-month interval.

Engraftment rates related to busulphan and cyclophosphamide dosages for displacement bone marrow transplants in fifty children.

The use of busulphan and cyclophosphamide permitted engraftment in 44 of 49 children receiving 'displacement' bone marrow transplants. Three patients who received T-cell-depleted marrow cells from HLA-haploidentical donors failed to engraft and other graft failures were due to inadequate induction dosage. Our standard schedule comprises busulphan 80 mg/m2/day x 4 days (adjusted if necessary to a minimum of 4 mg/kg/day or a maximum of 5 mg/kg/day) followed by cyclophosphamide 2 g/m2/day x 4 days but reduced so as not to exceed 75 mg/kg/day, a maximum dose preferred for patients with full marrows (e.g. those with thalassaemia major). Of 21 recipients of mixed lymphocyte culture (MLC)-negative donor marrow cells with full engraftment at 100 days, there were three late rejections. Of patients transplanted with marrow from MLC-positive donors, one had late rejection after cyclosporin A toxicity had necessitated withdrawal of the drug at day + 146 but six other patients, whose cyclosporin A was stopped routinely 1 year, remain well with full grafts. Ten patients died as a result of graft-versus-host disease. We are therefore exploring new approaches to T-cell depletion and storing autologous marrow for use in the event of graft failure. If necessary, a second transplant with busulphan and cyclophosphamide is best performed at 3 months after full recovery of the host. We conclude that elective transplants can be performed successfully in children with normal immune function without the need for irradiation.

Transmission of toxoplasmosis by bone marrow transplant associated with Campath-1G.

A 12-year-old boy in third remission acute lymphoblastic leukaemia was given a mismatched transplant from his mother. He suffered prolonged neutropenia and pyrexia which was only finally diagnosed as toxoplasmosis using molecular biology methods and by his response to appropriate treatment. This was probably transmitted by bone marrow transplant since maternal immune T cells were removed by the use of Campath-1G and treatment with cyclosporin A probably prevented his IgM immune response and impeded the diagnosis.

Incidence and pathogenesis of megaloblastic erythropoiesis in multiple myeloma.

Intermediate megaloblastic changes occurred in six (19%) of 32 patients with multiple myeloma and trivial megaloblastic changes in a further ten (31%). Folate deficiency was the predominant cause of these changes and in at least two patients was sufficiently severe to contribute to anaemia. Folate deficiency appeared to be due to excess folate utilization by the tumour and was related to the amount of paraprotein produced daily. Five of the 32 patients had subnormal serum B(12) levels. Reduction in the serum B(12) level was related to the reduction in the normal circulating immunoglobulins and occurred despite normal B(12) absorption. Possible explanations for this finding are discussed.

Crossreaction of antilymphocyte globulin with human granulocyte colony-forming cells.

Clinical preparations of horse antilymphocyteglobulin (ALG) were found to inhibit human bone marrow granulocyte colony growth. This effect was enhanced by complement and was dose dependent, being almost complete at ALG concentrations of 100 microgram/ml. Inhibition was a property of ALG but not of normal horse globulin. However, short incubation of ALG with bone marrow cells occasionally stimulated colony growth and normal horse globulin regularly stimulated it. Three hours' incubation of bone marrow cells with ALG was needed to produce consistent colony inhibition, which was measurable as a reduction in the expected number of colonies and as a fall in the colony: cluster ratio of surviving cell aggregates. Absorption of ALG on acute myeloid leukaemia blast cells removed the inhibiting property of the ALG while preserving its lymphocytotoxic action. Serum from two patients receiving ALG treatment inhibited colony growth for up to 48 hours after ALG administration. The results suggest the presence in ALG of antibodies specifically cytotoxic to myeloid stem cells which may relate to its myleosuppressive properties in vivo, and also indicate that it should be possible to remove antimyeloid antibodies from ALG by absorption. The use of such purified ALG would have advantages in clinical bone marrow transplantation.

Value of serum C-reactive protein measurement in the management of bone marrow transplant recipients. Part I: Early transplant period.

Serum C-reactive protein concentrations were measured serially during the early transplant period in 68 bone marrow recipients transplanted for leukaemia (34), chronic granulocytic leukaemia (2), severe aplastic anaemia (6), and various inborn errors of metabolism (26). There were 116 clearly documented episodes of infection or acute graft versus host disease or both. Serum C-reactive protein concentrations in patients with viral (11) or fungal infection (6) were normal or only slightly raised. In 32 patients with isolated acute graft versus host disease, only three (10%) showed serum C-reactive protein concentrations above 40 mg/l. Values greater than 40 mg/l were strongly suggestive of bacterial infections and values above 100 mg/l were seen only in patients (43) with bacterial infections with or without acute graft versus host disease. These findings suggest that serum C-reactive protein concentrations are valuable both for diagnosis and monitoring of such infections.

Value of serum C-reactive protein measurement in the management of bone marrow transplant recipients. Part II: Late post-transplant period.

Seventeen bone marrow recipients transplanted for acute leukaemia (8), chronic leukaemia (1), severe aplastic anaemia (3), and various inborn errors of metabolism (5) had 22 episodes of documented infection in the late (greater than 3 months) post-transplant period. Serum C-reactive protein concentrations were considerably increased in patients with bacterial infections, but not in those with viral or fungal infections. Serum C-reactive protein values were normal in 20 patients transplanted for acute leukaemia (12), chronic leukaemia (1), severe aplastic anaemia (2), and various inborn errors of metabolism (5) who had active chronic graft versus host disease but no evidence of infection. These findings indicate that serum C-reactive protein concentrations are useful in the diagnosis and monitoring of bacterial infections even in the presence of chronic graft versus host disease.

Increased serum IgE concentrations during infection and graft versus host disease after bone marrow transplantation.

Serum IgE concentrations estimated in 25 bone marrow transplant recipients during episodes of infection or graft versus host disease, or both, were raised not only in some patients with acute graft versus host disease but also in many patients with infection. Raised values were not seen in chronic graft versus host disease. The routine estimation of serum IgE in bone marrow transplant recipients had minimal value because of the lack of specificity of the IgE response.

Cytokine gene expression in skin and lymphoid organs in graft versus host disease.

AIM: To determine if human graft versus host disease (GvHD) is associated with any detectable change in cytokine gene expression in the skin and lymphoid organs. METHODS: Reverse transcriptase and the polymerase chain reaction were used to amplify mRNA for interleukins-1 (IL-1), -2 (IL-2), -4 (IL-4) and -6 (IL-6), IL-2 receptor (IL-2R), tumour necrosis factors alpha (TNF-alpha) and beta (TNF-beta), gamma interferon (IFN gamma) and granulocyte macrophage colony stimulating factor (GM-CSF) in frozen punch biopsy specimens of skin and necropsy samples of skin, lymph node, and spleen. RESULTS: No cytokine mRNA was detected in the punch biopsy specimens except weak signals for IL-6 and IL-1 and GM-CSF in two normal donors and IL2-R in one patient with GvHD. In samples of skin taken at necropsy, however, significant quantities of mRNA for TNF-alpha, TNF-beta, and IL-4 were detected in patients who had or had had GvHD in contrast to those without the disease whose skin lacked mRNA for these products but contained detectable quantities of IL-1, IL2-R, IL-6 and GM-CSF. There seemed to be a reciprocal relation between TNF-alpha and IL-4. In necropsy samples of lymph node and spleen a pattern of cytokine production similar to that in the skin was observed with a preponderance of TNF-alpha, TNF-beta and IL-4 in patients with GvHD and GM-CSF and IL-6 in those without the disease. CONCLUSIONS: The local synthesis of these molecules would explain many of the morphological and immunohistological features of GvHD. The failure to detect TNF-alpha, TNF-beta, and IL-4 in skin biopsy specimens exhibiting GvHD is probably due to their small size but further investigations are required.

Multiwell cap assay: a simple objective method for the assessment of leukocyte locomotion in vitro.

A simple method for evaluating leukocyte locomotion in vitro has been developed and validated for several chemoattractants. The multiwell cap assay (MWCA) comprises chambers constructed from readily available disposable plastics and is quickly assembled, permitting large experimental protocols. Leukocytes which have migrated through a micropore filter are recovered and counted electronically yielding a precise, objective result. Coefficients of variation are approximately 6%.

Overestimation of monoclonal immunoglobulin by immunochemical methods.

A comparison of paraprotein estimation by scanning densitometry with corresponding immunoglobulin concentration measured by rate immunonephelometry, fixed-time immunonephelometry and kinetic immunoturbidimetry using a large number of sera has indicated overestimation of the immunoglobulin by liquid phase immunoassays in the presence of a monoclonal component. The effect was seen with all three major immunoglobulin isotypes but was most marked with IgM paraproteins when measured by kinetic nephelometry. The overestimations could not be explained by presence of rheumatoid factors or immune complexes in the serum samples, or by non-parallelism, over a range of dilutions, of the reaction of sample and calibrant with the antiserum. The source and species of antiserum used was a major contributory factor.

An evaluation of the Hyland laser nephelometer PDQ system for the measurement of immunoglobulins.

The Hyland laser nephelometer PDQ system for the assay of specific proteins is described. The results of evaluating the system to measure immunoglobulins IgA, IgG, and IgM are summarised. Within-batch and between-batch precision, accuracy, reliability, and safety are discussed. This instrument represents an important development in the immunochemical assay of proteins in clinical medicine. The speed, precision, and convenience of this new generation of discrete nephelometric analysers make such systems attractive to the clinical chemist.

Regression of the radiological changes of Gaucher's disease following bone marrow transplantation.

Seven children with Type I Gaucher's disease have been treated with bone marrow transplantation (BMT). One patient died from the complications of BMT. In five patients computed tomography (CT) of the femora showed initially high attenuation in the marrow, returning towards normal after successful BMT. One of these patients also showed clearing of pulmonary infiltrates following treatment. In the first patient in the series no pre-transplant CT measurements were carried out but plain radiographs have demonstrated a return to normal shape of her femora 21/2 years after BMT. It is hoped that CT of the marrow cavities may provide a simple, non-invasive method for monitoring progress following BMT.

Chylous effusions due to neuroblastoma resolved by intrapleural chlorambucil coupled to father's antibodies.

A 2-year-old boy presented with a stage IV neuroblastoma and, despite surgical reduction of the tumor mass followed by chemotherapy and radiotherapy, he developed bilateral chylous pleural effusions. These proved resistant to traditional methods of treatment but were resolved with minimal discomfort to the child by intrapleural injections of chlorambucil chemically bound to antibodies raised by immunizing his father with irradiated tumor cells.