Functional characterization of canine lymphocyte subsets

Summary Functional characterization of subsets of T lymphocytes is essential for transplantation studies in dogs, as it is in other species. We studied the function of T cells separated by two mouse monoclonal antibodies recognizing complementary subsets — an antibody directed to canine T cells (MdT-P1) with an up-regulating function, and an antibody directed to human CD 8 (MT811) that cross-reacts with down-regulating canine T cells. Immunorosetting with sheep red blood cells and Percoll gradient allowed us to study depleted and enriched fractions. Their function was tested in mixed lymphocyte culture (MLC), cell-mediated cytotoxicity (CML), and coculture with B cells in a hemolytic plaque assay (PFC). In MLC, MdT-P1-positive cells showed a high proliferative response, and MT811-positive cells responded poorly to allogeneic cells. Vice versa, MT811-negative cells responded strongly, and MdT-P1-negative cells were poor responders but strong stimulators. Effector cells of CML were separated following 8 days of culture and prior to mixing with target cells. Enriched and depleted fractions with either antibody showed low cytotoxic activity as compared with unseparated cells. When added to unseparated effector cells MT811-positive cells suppressed cytotoxicity. B cells were obtained by resetting with staphylococcal protein A (SPA). Their immunoglobulin production was studied following 6 days of culture stimulated by pokeweed mitogen in a reverse hemolytic plaque assay. Again, MT811-positive cells added to the culture suppressed, and MT811-negative cells enhanced immunoglobulin production. In conclusion, immunorosetting with two monoclonal antibodies allowed us to distinguish subpopulations of canine T cells with up-regulating (helper/inducer) from those with down-regulating (suppressor) activity.This work was supported by theWilhelm-Sander-Stiftung     

Adoptive immunotherapy in chimeras with donor lymphocytes

Allogeneic stem cell transplantation has a well-defined indication in the treatment of hematological malignancies. The beneficial immune effect of allogeneic marrow transplantation has long been known, but only recently have methods been developed to separate the graft-versus-leukemia (GVL) effect from graft-versus-host disease (GVHD). Animal experiments have shown that lymphocytes from the marrow donor can be transfused without causing severe GVHD if stable chimerism and tolerance is established. First clinical studies have been preformed in patients with recurrent chronic myelogenous leukemia. In these patients complete molecular remissions were induced that persist without further maintenance treatment. These results have been confirmed in larger multicenter studies in Europe and the USA. The best results were obtained in chronic myelogenous leukemia (CML); repeated successes have been reported in relapsing acute myeloid leukemia (AML), myelodysplastic syndromes and multiple myeloma (MMY), and rare responses were reported for acute lymphoid leukemia. Contrary to animal experiments GVHD has been observed in human patients although to a lesser extent than expected in transplants not given immunosuppression. Secondly myelosuppression has been observed in patients treated with relapsing CML. In CML the incidence of GVHD could be reduced by depleting CD8+ T cells from the donor lymphocyte concentrate. Alternatively only small numbers of T lymphocytes can be transfused and in the case of failing responses, the numbers of donor lymphocytes may be increased. Results in recurrent AML have been improved by the use of low-dose cytosine arabinoside, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor mobilized blood cells as compared to lymphocytes only. In MMY the response rate is higher than in AML, but the remissions are of limited duration in most patients. Several protocols have been designed to include preemptive donor lymphocyte transfusion in patients with a high relapse risk after transplantation. Problems remain to avoid chronic GVHD and to circumvent the immune escape mechanisms of leukemia.     

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single syn1-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this syn1-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding anti1 isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the syn1- and anti1-mouse AChE complexes at 2.45- to 2.65-A resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the syn1 isomer induces large and unprecedented enzyme conformational changes not observed in the anti1 complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template.     

Inorganic phosphate treatment of nephrolithiasis

We report the results of long-term inorganic phosphate therapy in 47 patients suffering from recurrent calcium-containing urinary calculi. Forty-five patients had hypercalciuria before treatment. Phosphate therapy had no effect on urinary calcium excretion or stone passage but did appear to reduce the need for lithotomy. The stone passage-removal rate in the 5 years before therapy was 1.3 per patient per year and fell to 0.8 in the years of treatment (mean 5.5 years). The lithotomy rate fell from 0.12 per patient per year to 0.04 with treatment (p < 0.01). Patients who were free of stones at the start of the therapy fared best. Significant decreases in serum alkaline phosphatase were noted in many patients, possibly a sign of healing subclinical osteomalacia.     

Autoantibodies to the islet antigen ICA 69 occur in IDDM and in rheumatoid arthritis

Summary Islet cell antigen (ICA) 69 is a newly-recognized islet cell antigen to which autoantibodies have been observed in prediabetic relatives of patients with insulin-dependent-diabetes mellitus (IDDM). Here we extend the earlier analysis of ICA 69 antibodies to patients with recent-onset IDDM and to patients with other immune-mediated diseases. ICA 69 antibodies were determined by Western blot using an affinity purified recombinant fusion protein of ICA 69 and maltose binding protein. ICA 69 antibody quantities were determined as titres using a titration curve of a standard serum as reference. Mean logarithmic ICA 69 antibody titres were 3.4 (±1.4) in 99 patients with acute IDDM compared to 2.8 (±0.9) in 49 healthy blood donors (p<0.001). A higher mean ICA 69 antibody titre of 4.1 (±0.8) was observed in 16 patients with rheumatoid arthritis in comparison to acute IDDM (p<0.01) and healthy control subjects (p<0.001). The percentage of sera with ICA 69 antibody titres above the 2 SD level of normal subjects was 21% in IDDM, 31% in rheumatoid arthritis and 6% in healthy blood donors. None of the patients with autoimmune thyroid disease (n=20), inflammatory bowel disease (n=9) or multiple sclerosis (n=7) had elevated ICA 69 antibodies. In IDDM, presence of ICA 69 antibodies persisted and the titre remained the same over 18 months of follow-up. The relationship of ICA 69 antibodies to islet cell antibodies (ICA) or insulin autoantibodies (IAA) was tested. The production of ICA 69 antibodies was not associated in diabetic patients with the presence of any of the two other autoantibodies. In conclusion, this study describes ICA 69 antibodies in acute IDDM and finds them to be independent of other islet autoantibodies. In addition ICA 69 is a target of humoural autoimmunity not only in IDDM but also in rheumatoid arthritis.Abbreviations IDDM Insulin-dependent diabetes mellitus - ICA islet cell antibodies - IAA insulin autoantibodies - RA rheumatoid arthritis - RF rheumatoid factor - GAD 65 glutamic acid decarboxylase - SMS stiff-man syndrome     

Clinical studies with computer-assisted initial lidocaine therapy

A randomized prospective study compared achievement and maintenance of therapeutic plasma concentrations in patients receiving computer-assisted (CA) initial lidocaine hydrochloride therapy, designed pharmacokinetically to achieve and maintain a chosen plasma concentration, v conventional lidocaine therapy (CT). A separate audit of outcome was also conducted. The CA regimens provided more effective concentrations in the first hour than did CT, 2.65 v 1.5 micrograms/mL average. In the audit, ventricular fibrillation occurred in two of 78 CA v eight of 78 CT patients. Dosage adjustments were required in two CA patients v 33 CT patients. The CA therapy improved therapeutic precision, reduced dosage adjustments, and may have improved safety during initial lidocaine therapy before fitting to plasma concentration data for subsequent feedback. An improved clinical computer program now also fits to plasma concentration data. It is accessed and used routinely by hospitals over an international time-sharing network.     

Non-specific viral infections as possible synchronising events of the manifestation of type 1 diabetes

Four cases of simultaneous manifestation of Type 1 diabetes in two members of the same household are reported. In all cases, a flu-like infection preceded diabetes onset. Surprisingly, despite simultaneous development of insulin dependency, insulin requirements were strikingly different at 3 months in all cases. These observations suggest that increased insulin resistance during infection may cause insulin deficiency in individuals with widely varying residual beta cell activity.     

Investigation of the Relationship Between Prostate Cancer and MSMB and NCOA4 Genetic Variants and Protein Expression

Two genome‐wide association studies (GWAS) identified the β‐microseminoprotein (MSMB) promoter SNP, rs10993994:C>T, as significantly associated with prostate cancer (PC) risk. Follow‐up studies demonstrate that the variant allele directly affects expression of the MSMB‐encoded protein, PSP94, and also suggest that it affects mRNA expression levels of an adjacent gene, NCOA4, which is involved in androgen receptor transactivation. In a population‐based study of 1,323 cases and 1,268 age‐matched controls, we found the NCOA4 SNP, rs7350420:T>C, was associated with a 15% reduction in PC risk, but the association was not significant after adjustment for the rs10993994:C>T genotype. Tumor tissue microarrays of 519 radical prostatectomy patients were used to measure PSP94 and NCOA4 protein expression. Taken together, these data confirm that the rs10993994:C>T variant allele is associated with decreased PSP94 expression, and the association is stronger in tumor compared to normal prostate tissue. No association was observed between rs10993994:C>T and NCOA4 expression, and only moderate associations were seen between two NCOA4 SNPs, rs10761618:T>C and rs7085433:G>A, and NCOA4 protein expression. These data indicate that the increase in PC risk associated with rs10993994:C>T is likely mediated by the variant's effect on PSP94 expression; however, this effect does not extend to NCOA4 in the data presented here.