Bacterial joint infections in England and Wales: analysis of bacterial isolates over a four year period

Data from 1158 cases of septic arthritis reported to the Public Health Laboratory Service (PHLS) Communicable Disease Control Centre (CDSC) from England and Wales over a 4 yr period (January 1990 December 1993) are presented. Reports where a bacterial organism was isolated from synovial fluid, or where an organism was isolated from blood cultures where a diagnosis of septic arthritis was reported, were examined. Reports of infection were more common in children (12.7% of infections were in the under 10 age group) and the elderly (54.7% aged 60 or over), and were higher in males in all age groups except in the elderly. The most common causative organisms remain staphylococcal and streptococcal species, comprising 40.6% (470) and 28% (324) of cases, respectively. The most common streptococci seen were Streptococcus pneumoniae and Lancefield group A beta-haemolytic Streptococcus organisms, 60.8% (197/324), although group B, C and G organisms accounted for 33.6% of streptococcal isolates (109/324). Haemophilus influenzae septic arthritis is not exclusive to children as 23.2% (16- 69) of cases occurred over the age of 15. A total of 48% (635) of isolates were identified from both synovial fluid and blood cultures, 32.6% (378) from joint fluid alone and 12.5% (146) from blood cultures. Although this study excludes cases of septic arthritis where no organism was isolated, it presents important bacteriological information from a large number of isolates from England and Wales over a 4 yr period. Risk factors identified include a joint prosthesis, joint disease/connective tissue disorder. immunosuppression and diabetes.      

Human CD4 lymphocytes specifically recognize a peptide representing the fusion region of the hybrid protein pml/RAR alpha present in acute promyelocytic leukemia cells

Fusion proteins present in leukemic cells frequently contain a new amino acid at the fusion point. We tested whether a peptide (BCR1/25) encompassing the fusion region of the hybrid molecule pml/RAR alpha, which is selectively expressed by acute promyelocytic leukemia (APL) cells, can be recognized by human T lymphocytes in vitro. CD4+ lymphocytes, at both polyclonal and clonal level, recognized peptide BCR1/25 in an HLA-DR--restricted fashion on presentation by autologous antigen-presenting cell (APC) or by APC expressing the HLA-DR11 restricting molecule. Control peptides corresponding to the normal pml and RAR alpha proteins were not recognized. One clone (DEG5) also exerted a high and specific cytotoxicity against autologous cells pulsed with BCR1/25. The autologous DE LCL containing a transduced pml/RAR alpha fusion gene and expressing a bcr1 type of the pml/RAR alpha hybrid protein induced the proliferation of DE anti-BCR1/25 T cell clones. It is concluded that the bcr1 type-pml/RAR alpha fusion protein of APL contains an antigenic site, absent from the normal parent molecules and recognized by human CD4+ lymphocytes.     

Retroviral-mediated gene transfer in human bone marrow cells growth in continuous perfusion culture vessels

Hematopoietic stem cell gene therapy holds the promise of being able to treat a variety of inherited and acquired diseases of the hematopoietic stem cell. However, to date, genetic modification of the human hematopoietic stem cell has been relatively inefficient. Here, we report the results of using a bioreactor system to expand hematopoietic cells after a brief retrovirus infection using a high titer, replication defective virus encoding for murine CD18. The retrovirus transduced culture continued to produce genetically modified hematopoietic progenitors for up to 6 weeks, the duration of the culture period. Up to one-third of the long-term culture initiating cell (LTC-IC) are genetically modified by the culture conditions. Murine CD18 can be expressed on the cell surface of up to 20% of the mature cells generated by the culture system, suggesting that clinically significant levels of gene transfer may be occurring. These results demonstrate the feasibility of using continuous perfusion bioreactors as a method of efficiently modifying human hematopoietic stem cells.     

The accumulation of p53 abnormalities is associated with progression of mucosa-associated lymphoid tissue lymphoma

The genetic mechanisms underlying the genesis of low-grade mucosa- associated lymphoid tissue (MALT) lymphomas and their transformation into high-grade lymphoma are poorly understood. p53 inactivation, commonly caused by mutation and allele loss, has been shown to play an important role in the early development and/or the late disease progression of many human tumors including lymphoid malignancies and, thus, may also be important in MALT lymphomagenesis. We examined 75 cases (48 low grade and 27 high grade) of MALT lymphoma for p53 allele loss and mutation as well as protein accumulation. DNA samples prepared from microdissected cell populations were used for the detection of p53 gene abnormalities. Loss of heterozygosity (LOH) of the gene was detected by polymerase chain reaction-based analysis of p53 CA repeat polymorphism, whereas p53 mutation was studied by single-strand conformation polymorphism analysis and direct sequencing. p53 expression was assessed by immunostaining with CM1 polyclonal antibody. p53 allele loss and mutation, which resulted in the alteration in the amino acid sequence, were found in both low-grade (LOH, 3 of 44 [6.8%]; mutation, 9 of 48 [18.8%]) and high-grade (LOH, 6 of 21 [28.6%]; mutation, 9 of 27 [33.3%]) MALT lymphomas, particularly in the latter group. p53 staining was not observed in any low-grade tumors but in 6 high-grade cases that harbored missense mutations. There were also differences in the extent of p53 abnormalities, between low- and high- grade tumors. Of the 11 low-grade tumors showing p53 abnormalities, only 1 tumor showed the concomitance of p53 mutation and allele loss, whereas in high-grade tumors, 6 of 9 affected cases displayed both p53 mutation and allele loss. Our results suggest that p53 partial inactivation may play an important role in the development of low-grade MALT lymphomas, whereas complete inactivation may be associated with high-grade transformation.     

Human umbilical vein endothelial cells display high-affinity c-kit receptors and produce a soluble form of the c-kit receptor

Stem cell factor (SCF) is a hematopoietic growth factor produced by fibroblasts and endothelial cells that stimulates the growth of primitive hematopoietic cells. SCF triggers cell growth by binding to the c-kit receptor. Because endothelial cells can respond to certain hematopoietic growth factors, we tested human umbilical vein endothelial cells for display of the c-kit receptor and examined the effect of SCF on endothelial cell proliferation, adhesion molecule expression, and production of tissue factor. Quantitative binding experiments with 125I-SCF showed both high-affinity (Kd = 42 pmol/L) and low-affinity (Kd = 1.7 nmol/L) c-kit receptors. There were approximately 1,100 high-affinity c-kit receptors, and 5,400 low- affinity c-kit receptors per endothelial cell. Enzyme immunoassays showed that endothelial cells released soluble c-kit receptor and SCF. The transmembrane form of SCF was detected by indirect immunofluorescence analysis using monoclonal or polyclonal anti-SCF receptor antibodies. The addition of SCF (100 ng/mL) did not alter endothelial cell proliferation over a 7-day period. Similarly, there was no change in the release of tissue factor or expression of inducible endothelial adhesion molecules (intercellular adhesion molecule-1, endothelial-leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1) measured by enzyme-linked immunosorbant assay at 4 and 24 hours after SCF addition. The neutralizing anti-c-kit receptor monoclonal antibody SR-1 blocked binding of 125I-SCF to the c- kit receptor by 98% but did not alter endothelial cell proliferation or adhesion-molecule expression. c-kit receptors were also detected on adult endothelial cells lining small blood vessels in normal human lymph nodes. These data indicate that normal human endothelial cells produce SCF and show high-affinity c-kit receptors that have the capacity to dimerize. The lack of response to exogenous SCF may be because of intracellular activation of the c-kit receptor via autocrine production of SCF. Alternatively, SCF and c-kit may play a role other than stimulation of proliferation, adhesion-molecule display, or tissue factor production by endothelial cells. The production of soluble c-kit receptors by normal human endothelial cells may serve to regulate the bioactivity of SCF within the bone marrow microenvironment.     

Platelet adhesion to collagen and endothelial cell matrix under flow conditions is not dependent on platelet glycoprotein IV

Platelet membrane glycoprotein IV (GPIV) is a cell-surface glycoprotein that has been proposed as a receptor for collagen. Recently, it has been shown that platelets with the Naka-negative phenotype lack GPIV on their surface, whereas donors with this phenotype are healthy and do not suffer from hematologic disorders. In this study, we compared Naka- negative platelets with normal platelets in adhesion to collagen types I, III, IV, and V and the extracellular matrix of endothelial cells (ECM) under static and flow conditions. No differences in platelet adhesion and subsequent aggregate formation on the collagens types I, III, and IV were observed under static and flow conditions. Adhesion of both homozygous and heterozygous Naka-negative platelets to collagen type V was strongly reduced under static conditions. Collagen type V was not adhesive under flow conditions. No difference in platelet adhesion to ECM was observed, which suggests that GPIV is not important in adhesion to subendothelium, for which ECM may serve as a model. These results indicate that GPIV is not a functional receptor for collagen under flow conditions.     

Depressed functional and phenotypic properties of T but not B lymphocytes in idiopathic thrombocytopenic purpura

Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which the abnormality in cellular immunity has remained only vaguely defined. Previously we have shown that patients with ITP in its active phase have abnormal T cell subsets. We then examined the phenotypes of T and B lymphocytes in an additional 28 patients with ITP and 32 age- and sex-matched normal controls and compared the lymphocytes' capacity to respond to polyclonal T, T cell-dependent B, and B cell mitogens. Blastogenesis to optimal (5.0 micrograms/mL) and suboptimal (0.5 microgram/mL) concentrations of the polyclonal T cell mitogens were markedly depressed in patients compared with normal controls (P less than .0005). Similarly, a severe depression in response was noted with the polyclonal T cell-dependent B cell mitogen (P less than .000001). No difference was seen, however, with the polyclonal B cell mitogen. The proportions of pan-T and T helper/inducer lymphocytes were significantly depressed (P less than .005 and P less than .000005 respectively), and the T suppressor/cytotoxic lymphocytes increased (P less than .02) in patients relative to controls. But there was no difference in the proportion of B lymphocytes or in their functional response. The abnormal cellular immunity appears to be due to a defect in the T lymphocyte population without involvement of the B lymphocytes.     

Infrequent alterations of the RAR alpha gene in acute myelogenous leukemias, retinoic acid-resistant acute promyelocytic leukemias, myelodysplastic syndromes, and cell lines

Retinoids are important regulators of cell growth and differentiation in vitro and in vivo and they exert their biologic activities by binding to nuclear retinoic acid receptors (RARs; alpha, beta, and gamma) and retinoid X receptors (RXRs; alpha, beta, and gamma). All- trans retinoic acid (RA) induces complete remission in patients with acute promyelocytic leukemia (APL) presumably by binding directly to RAR alpha of APL cells. Leukemic blasts from APL patients initially responsive to RA can become resistant to the agent. HL-60 myeloblasts cultured with RA have developed mutations of the ligand-binding region of RAR alpha and have become resistant to RA. Furthermore, insertion of an RAR alpha with an alteration in the ligand-binding region into normal murine bone marrow cells can result in growth factor-dependent immortalization of the early hematopoietic cells. To determine if alterations of the ligand binding domain of RAR alpha might be involved in several malignant hematologic disorders, the mutational status of this region (exons 7, 8, and 9) was examined in 118 samples that included a variety of cell lines and fresh cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including 20 APL patients, 5 of whom were resistant to RA and 1 who was refractory to RA at diagnosis, using polymerase chain reaction-single- strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, 7 of the 20 APLs were studied for alterations of the other coding exons of the gene (exons 2 through 6). No mutations of RAR alpha were detected. Although the sensitivity of PCR-SSCP analysis is less than 100%, these findings suggest that alterations of RAR alpha gene are rare and therefore other mechanisms must be involved in the onset of resistance to retinoids and in the lack of differentiation in disorders of the myeloid lineage.     

Nodular lymphocyte predominance Hodgkin's disease: a monoclonal or polyclonal B-cell disorder?

Nodular lymphocyte predominance Hodgkin's disease (NLPHD) is characterized by the presence of atypical putatively neoplastic cells (L & H cells) with a B-cell phenotype. A proportion of patients with NLPHD develop a simultaneous or subsequent large cell B lymphoma (LCL) that is thought to evolve directly from the L & H cells of NLPHD. However, the clonal nature of L & H cells remains controversial, and the relationship between NLPHD and complicating LCL has not been fully established. In an attempt to determine the clonality of L & H cells and to clarify the link between NLPHD and complicating LCL, we used polymerase chain reaction (PCR) to analyze 33 cases of NLPHD, including 15 cases with simultaneous or subsequent LCL, for clonal immunoglobulin (lg) heavy chain variable region (VH) gene rearrangements. PCR amplifications with consensus primers covering framework 2 or framework 3 to joining region were performed on paraffin-embedded tissue sections and, in 12 cases, on microdissection-enriched L & H cells. No clonal Ig rearrangements were detected. In eight of the 15 LCL, monoclonal IgVH regions were amplified, four of which were cloned and sequenced. Clone specific primers were designed based on the unique N region sequences. These allowed detection of LCL clones at a sensitivity up to 1,000 times greater than the consensus primers, as determined by dilution assays. However, no LCL clones were detected in the preceding NLPHD, including microdissection-enriched L & H cells. Our results suggest that populations of L & H cells do not carry monoclonal Ig rearrangements and provide no evidence for a clonal link between NLPHD and complicating LCL.     

Platelet adhesion to collagen type IV under flow conditions

Collagen type IV is a sheet-forming collagen and a major constituent of the vessel wall. To find out which conditions are important for platelet adhesion to collagen type IV, we performed perfusion studies with anticoagulated blood in parallel plate perfusion chambers. The role of divalent cations was investigated by using plasmas with variable concentrations of Mg2+ and Ca2+ ions. When Mg2+ concentration was decreased from 2.00 mmol/L to 0.25 mmol/L at a fixed Ca2+ concentration of 1.25 mmol/L, platelet coverage on the collagen type IV surface decreased from 22.8% +/- 1.8% (n = 4) to 4.6% +/- 0.6% (n = 4) at a shear rate of 1,600 s-1. Also, platelet aggregate formation on collagen type IV was strongly impaired. A monoclonal antibody against the glycoprotein (Gp) Ib receptor and von Willebrand factor (vWF)- depleted plasma reduced the platelet coverage to collagen type IV to, respectively, 10% and 45% of the control value. Electron microscopy showed that vWF was only present between platelets and between the platelet and the collagen type IV surface, but did not bind elsewhere to collagen type IV. These data indicate that collagen type IV is a reactive collagen for platelets. Differences in physiologic plasma magnesium concentrations may in part explain the differences in platelet reactivity to collagen type IV between individuals, and perhaps contribute to differences in the risk for thrombosis.