Morphology of bacteriophage P22 as seen in thin sections of pelleted phage.

The tails of adsorbed P22 in thin section appear longer than tails seen on negatively stained phage. Phage were pelleted and sectioned to determine tail length on free phages. The tail length of both adsorbed and unadsorbed phage in thin section were nearly identical, approximately 240 Å. This represents a tail length about one-third longer than that observed in negatively stained specimens. Areas of phage pellets were in highly ordered arrays (paracrystalline arrays). The phage head in thin section is sliced into shapes that are consistent with the head being an icosahedron.     

Characterization of autoantigenic epitopes on platelet glycoprotein IIb/IIIa using random peptide libraries

Most patients with chronic immune thrombocytopenic purpura (ITP) have autoantibodies directed against the glycoprotein (GP) IIb/IIIa complex. We have used a filamentous phage library that displays random linear hexapeptides to identify peptide sequences recognized by these autoantibodies. Plasma antibody eluates from two patients were used to select for phage displaying autoantibody-reactive peptides. From patient ITP-1 (known to have two distinct autoantibodies), we identified anti-GPIIb/IIIa antibody-specific phage encoding the peptide sequences Arg-Glu-Lys-Ala-Lys-Trp (REKAKW) and Pro-Val-Val-Trp-Lys-Asn (PVVWKN). Patient ITP-2 bound phage encoding the hexapeptide sequence Arg-Glu-Leu-Leu-Lys-Met. Each phage showed saturable dose-dependent binding to immobilized autoantibody, and binding could be blocked with purified GPIIb/IIIa. Patient ITP-1 autoantibody recognition of phage encoding REKAKW could be blocked with a synthetic peptide derived from the GPIIIa cytoplasmic tail; however, the PVVWKN was not. Using sequential overlapping peptides from the GPIIIa cytoplasmic region, an epitope for ITP-1 was localized to the sequence Arg-Ala-Arg-Ala-Lys-Trp (GPIIIa 734-739). Inhibition studies using synthetic peptides showed that phage REKAKW and PVVWKN were recognized by distinct autoantibodies from patient ITP-1. To determine whether individual patients with ITP possessed autoantibodies that recognize similar antigenic determinants on GPIIb/IIIa, the three phage were tested for binding to five other ITP patient autoantibodies. The phage encoding the peptide PVVWKN was found to bind ITP-1 and one other patient autoantibody. This result suggests that ITP patients recognize a limited number of shared epitopes.     

CD40 ligand triggered interleukin-6 secretion in multiple myeloma

Previous studies have suggested that interleukin-6 (IL-6) may mediate growth of multiple myeloma (MM) in either an autocrine or paracrine growth mechanism. However, those molecules which can trigger IL-6 secretion either by tumor cells or non-MM marrow cells are not well characterized. In the present study, we have examined the expression and functional significance of CD40 on MM and plasma cell leukemia (PCL) cells and derived cell lines, as well as long-term bone marrow stromal cells (BMSCs) and derived cell lines. CD40 was expressed on the majority of MM cells (> 90%) and BMSCs (> 70%). Triggering via CD40 using NIH3T3 CD40 ligand transfectant (CD40LT) cells increased (> 30%) cell surface CD80, CD18, CD11a, CD11b, and CD11c expression on MM cell lines. Culture with either fresh or paraformaldehyde fixed NIH3T3 CD40LT cells upregulates IL-6 secretion in MM cells and MM-derived cell lines, as well as normal and MM bone marrow mononuclear cells (BMMCs), BMSCs, and BMSC lines; this effect can be specifically blocked by anti- CD40 monoclonal antibody (MoAb). BMMCs and BMSCs from patients with MM secreted significantly more IL-6 than those from healthy donors (n = 3, P < .001); moreover, after stimulation using CD40L, IL-6 secretion was fourfold greater (n = 3, P < .001) from MM BMMCs and BMSCs than from normal BMMCs and BMSCs. Myeloma (CD38+CD45RA-) cells and non-MM (CD38+CD45RA+, CD38-CD45RA+, and CD38-CD45RA-) BMMCs were separated by dual fluorescence cell sorting. The latter secreted fourfold more IL-6 than the former (n = 2, P < .001). Increased IL-6 secretion (up to 28- fold) and proliferation (Stimulation index 10) by CD38+CD45RA-MM cells was triggered by culture with NIH3T3 CD40LT cells. Finally, anti- CD40MoAb partially (30%) blocked tumor cell to BMSC adhesion-induced IL- 6 secretion. These studies support the view that CD40L may trigger IL-6 secretion by both MM cells and BMSCs and that IL-6-mediated autocrine and paracrine growth mechanisms may be possible in MM.     

Marrow transplantation with or without donor buffy coat cells for 65 transfused aplastic anemia patients

Sixty-five multiply transfused patients with severe aplastic anemia were given cyclophosphamide followed by grafts anemia were given cyclophosphamide followed by grafts from HLA-identical siblings. The effect of the administration of viable donor buffy coat cells following the marrow inoculum was evaluated with regard to graft rejection and survival. Results in 43 patients so treated are presented along with those in 22 concurrent patients given marrow alone. Most patients given buffy coat had positive in vitro tests of sensitization indicating a high risk for graft rejection, while all but one of the patients given marrow alone had negative tests. Thirty of the 43 (70%) patients given marrow and buffy coat are alive between 10 and 61 mo (median 36) after grafting; 4 died after graft rejection and 6 with acute or chronic graft-versus-host disease (GVHD). Eleven of the 22 (50%) patients given marrow alone are alive between 29 and 65 mo (median 52); 7 died after graft rejection and 3 with GVHD. The addition of buffy coat cell infusions to the marrow inoculum reduced the risk of rejection and increased survival in the currently reported transfused patients when compared to patients grafted before 1976. However, there was an increased risk of chronic GVHD. Recipients of marrow from female donors survived slightly better (73%) than recipients of male marrow (58%).     

MECHANISMS OF HYPOPHOSPHATAEMIA IN ALCOHOLIC PATIENTS

The aim of our study was to determine the possible pathophysiological mechanisms of hypophosphataemia in a group of 127 patients admitted to hospital for alcohol-related causes. Blood and fresh urine specimens were taken to determine acid-base and electrolyte parameters. Thirty-seven patients (29.1%) had hypophosphataemia (serum phosphorus <0.77 mmol/l) with a range of serum phosphorus of 0.32-0.74 mmol/l. In 17 hypophosphataemic patients inappropriate phosphaturia (FEP04 >20%, TmPO₄/GFR<0.80 mmol/l) was evident, possibly due to hypomagnesaemia, metabolic acidosis, metabolic alkalosis, or a proximal tubular defect in phosphate transport. The causes of hypophosphataemia in the remaining 20 patients were alcohol withdrawal syndrome, respiratory alkalosis and diarrhoea. Patients with hypophosphataemia were more often found to have hypomagnesaemia and respiratory alkalosis than normophosphataemic patients. In conclusion, hypophosphataemia is frequently observed in alcoholic patients due to various pathophysiological mechanisms, such as inappropriate phosphaturia, increased phosphorus entry into cells and increased gastrointestinal loss of phosphate.     

Evaluation of maternal plasma creatine kinase activity as a marker of abnormal early pregnancy

We have tested the value of maternal plasma creatine kinase activity for diagnosing ectopic pregnancies obtained after in-vitro fertilization and embryo transfer. Plasma creatine kinase was assayed in 57 patients: 20 normal, 23 miscarriages and 14 ectopic pregnancies, for a total of 240 samples. All values were in the lower part of the normal range except only one in a miscarrying patient. A statistically significant difference was observed for a cut-off value of 45 IU/l between normal and ectopic pregnancies. However, for this cut-off point, the measurement of plasma creatine kinase activity had a sensitivity of 0.50 and a specificity of 0.76 for the diagnosis of ectopic pregnancy. The positive predictive value was 0.69. Creatine kinase activity measurements are thus of no practical value in this particular population, in which an early and specific marker of ectopic implantation would be of paramount interest. The association of human chorionic gonadotrophin (HCG) determinations and ultrasound scanning of the pelvis still remain the best paraclinical support for an early diagnosis of ectopic implantation.