Etoposide causes illegitimate V(D)J recombination in human lymphoid leukemic cells

Etoposide is one of the most widely used antineoplastics. Unfortunately, the same treatment schedules associated with impressive efficacy are associated with an increased risk of secondary acute myeloid leukemia (AML), which has prompted its withdrawal from some treatment regimens, thereby potentially compromising efficacy against the original tumor. Because etoposide-associated AML is characterized by site-specific illegitimate DNA recombination, we studied whether etoposide could directly cause site-specific deletions of exons 2 and 3 in the hprt gene. Human lymphoid CCRF-CEM cells were treated with etoposide for 4 hours, and DNA was isolated after subculturing. The deletion of exons 2 and 3 from hprt was assayed by a quantitative polymerase chain reaction (PCR) method. In the absence of etoposide treatment, the frequency of deletions of exons 2 and 3 was very low (5.05 x 10(-8)). After exposure to 10 mumol/ L etoposide, the frequency of the exon 2 + 3 deletion was increased immediately after and at 24 hours after etoposide treatment (65 to 89 x 10(-8)) and increased to higher levels (128 to 173 x 10(-8)) after 2 and 6 days of subculture (P < .001 overall). The frequency of the exon 2 + 3 deletion assessed at 6 days of subculture after 4 hours of 0, 0.25, 1, 2.5, 5, and 10 mumol/L etoposide treatment increased with etoposide concentration, ie, 5.05 x 10(-8), 89.2 x 10(-8), 108 x 10(-8), 142 x 10(-8), 163 x 10(-8), and 173 x 10(-8), respectively (P < .0001). Sequencing of a subset of amplified products confirmed the presence of DNA sequences at the breakpoints consistent with V(D)J recombination. By contrast, exon 2 + 3 deletions after etoposide treatment in the myeloid cell lines KG-1A and K562 showed no evidence of V(D)J recombinase in their genesis. We conclude that etoposide can induce the illegitimate site-specific action of V(D)J recombinase on an unnatural DNA substrate after a single treatment in human lymphoid cells.     

Intriguing issues of EGFR targeting in head and neck cancer

We have read the recent comprehensive review by Cruz et al.[1] regarding the targeting of receptor tyrosine kinases andtheir therapeutic perspectives in head and neck squamous cellcarcinomas (HNSCC). The major focus of this report was epidermalgrowth factor receptor (EGFR) biology and targeting. However,we feel     

Survival response of a human glioma cell line to hyperthermia associated with rhein.

The effect of association of hyperthermia with the anti-inflammatory drug rhein (RH), 4,5-dihydroxyanthraquinone-2-carboxylic acid, on the clonogenic activity of human glioma cells has been examined. RH inhibits neoplastic growth mainly through an ATP depletion, but thermal cell killing is not mediated by the drug-induced changes in the energy status of the cell. The analysis of the interaction between RH and hyperthermia, performed with the isobolar method, demonstrates an additivity of the response so that the effectiveness of the combined treatment is the result of two independent effects. Although the effect of this combination is purely additive, RH allows us to achieve a pre-established cell killing with exposure times at 42 degrees C, which is generally accepted to be clinically achievable. RH might, therefore, be employed to reduce the side effects of hyperthermia without impairing its therapeutic effectiveness.     

Myelin thickenings in val 102/fs null mutation of MPZ gene

Painful sensory neuropathies consist of a wide range of neuropathies that can involve large as well as small nerve fibres. Even if most cases remain of unknown cause, some of them may be associated with an underlying disorder such as diabetes, HIV, infections, amyloidosis, and Sjogren's syndrome. Since in some cases an autoimmune mechanism has been postulated, we investigated a panel of circulating autoantibodies including anti‐gliadin (AGA), anti‐endomysium (EmA), anti‐transglutaminase (tTGA) and anti‐nuclear (ANA) antibodies in the sera of patients with unexplained painful sensory neuropathies in order to identify other potentially treatable disorders. We tested the sera of 10 patients (4M; 6F) previously investigated for other causes of neuropathies, including anti‐nerve, onconeural, anti‐extractable nuclear, anti‐neutrophil cytoplasmic, anti‐thyroglobulin (TgA) and anti‐peroxidase (TPOA) antibodies. We found the presence of AGA positivity in 4 patients (40%), ANA in 7 (70%) and AGA + ANA in 4 (40%), two of whom were negative for celiac disease by gastrointestinal biopsy. None of the patients had EmA positivity. Three (30%) had TgA and TPOA and none had anti‐nerve or onconeural antibodies. Whether the presence of circulating autoantibodies in patients with unexplained painful neuropathy reflects an autoimmune involvement which may be amenable to immune therapy and not only to symptomatic treatment remains to be established.     

Proinflammatory Response of Human Osteoblastic Cell Lines and Osteoblast-Monocyte Interaction upon Infection with Brucella spp.

The ability of Brucella spp. to infect human osteoblasts and the cytokine response of these cells to infection were investigated in vitro. Brucella abortus, B. suis, B. melitensis, and B. canis were able to infect the SaOS-2 and MG-63 osteoblastic cell lines, and the first three species exhibited intracellular replication. B. abortus internalization was not significantly affected by pretreatment of cells with cytochalasin D but was inhibited up to 92% by colchicine. A virB10 mutant of B. abortus could infect but not replicate within osteoblasts, suggesting a role for the type IV secretion system in intracellular survival. Infected osteoblasts produced low levels of chemokines (interleukin-8 [IL-8] and macrophage chemoattractant protein 1 [MCP-1]) and did not produce proinflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor alpha [TNF-α]). However, osteoblasts stimulated with culture supernatants from Brucella-infected human monocytes (THP-1 cell line) produced chemokines at levels 12-fold (MCP-1) to 17-fold (IL-8) higher than those of infected osteoblasts and also produced IL-6. In the inverse experiment, culture supernatants from Brucella-infected osteoblasts induced the production of IL-8, IL-1β, IL-6, and TNF-α by THP-1 cells. The induction of TNF-α and IL-1β was largely due to granulocyte-macrophage colony-stimulating factor produced by infected osteoblasts, as demonstrated by inhibition with a specific neutralizing antibody. This study shows that Brucella can invade and replicate within human osteoblastic cell lines, which can directly and indirectly mount a proinflammatory response. Both phenomena may have a role in the chronic inflammation and bone and joint destruction observed in osteoarticular brucellosis.Brucella spp. are gram-negative facultative intracellular bacteria that infect domestic and wild animals and can be transmitted to humans, in whom they produce a debilitating and eventually chronic disease. The most common clinical features of human brucellosis are undulant fever, sweats, arthralgias, myalgias, lymphadenopathy, and hepatosplenomegaly (35). Osteoarticular brucellosis is the most common localization of active brucellosis, although its reported prevalence varies widely. The three most common forms of osteoarticular involvement are sacroiliitis, spondylitis, and peripheral arthritis (1, 15, 23, 28, 38).Brucellar arthritis is frequently polyarticular and usually affects knees, sacroiliac joints, shoulders, and hips (28). In some cases, brucellar arthritis may be destructive, with associated osteopenia and cartilage damage. Brucellar spondylitis, which is more destructive than arthritis and causes more serious complications than arthritis does (7), typically begins at the disco-vertebral junction but may spread to the whole vertebrae and to adjacent vertebral bodies (29, 50).While the clinical and imaging aspects of osteoarticular brucellosis have been described widely, the pathogenic mechanisms of joint and bone disease caused by Brucella have not been investigated at the molecular and cellular levels. Regarding brucellar arthritis, a septic form and a reactive form have been proposed (15). The septic form is supported by the isolation of Brucella spp. from synovial fluid or tissue.In osteoarticular infections by pathogens such as Staphylococcus aureus and Mycobacterium tuberculosis, bone and joint damage results mainly from the inflammatory reaction elicited by the infection. In the mouse model of S. aureus arthritis, polymorphonuclear leukocytes and macrophages are seen in the synovial tissue early in the infection (5, 44). Similarly, an infiltrate of highly activated polymorphonuclear leukocytes has been observed in posttraumatic infectious osteomyelitis in humans (45). These cells produce not only proinflammatory cytokines and chemokines but also a series of tissue-degrading enzymes, including metalloproteinases, which can contribute to joint and bone destruction (13, 48). High levels of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) are detected in the synovial fluid of patients with bacterial arthritis (34, 40). Increased local levels of TNF-α mRNA have also been detected in a rat model of osteomyelitis (27). These cytokines stimulate the release of proteases by inflammatory cells (41). In addition, TNF-α and IL-1β, together with IL-6, stimulate osteoclast differentiation and bone resorption in a synergistic fashion (19, 26). In human brucellar arthritis, synovial fluid usually presents an increased leukocyte count, and the synovial membrane frequently exhibits a nonspecific inflammatory change (28).Given the central role of inflammatory cells in bone and joint destruction in osteomyelitis and arthritis, the recruitment and activation of these cells are of utmost importance for the development of these pathological conditions. Besides their role in bone formation, osteoblasts have also been shown to respond to bacterial infection or bacterial products by secreting proinflammatory cytokines, such as IL-6 and IL-12 (2, 20), and chemokines, such as macrophage chemoattractant protein 1 (MCP-1), IL-8, IP-10, and RANTES (4, 31, 47, 49), which recruit macrophages, neutrophils, and T lymphocytes. Overall, these data point to an active role of osteoblasts in the immune responses elicited during osteoarticular infections.Staphylococcus aureus and Mycobacterium tuberculosis, which are common etiological agents of osteoarticular infections, can infect human osteoblasts in vitro (11, 12, 22, 46, 47). The intracellular persistence of these bacteria in bone cells may facilitate disease progression by protecting these organisms from extracellular host defenses and antibiotic therapy and may help to explain the recurrent nature of osteomyelitis (12). Brucella spp. are known to survive and replicate within mononuclear phagocytes (32) and also in nonphagocytic cells, including epithelial cells and fibroblasts (37). In contrast, there are no data on invasion and/or intracellular replication of Brucella spp. within osteoblasts.In the present study, we investigated whether Brucella spp. can infect and survive within human osteoblastic cell lines and whether this infection elicits the secretion of proinflammatory cytokines and chemokines that might be involved in the osteoarticular manifestations of brucellosis. Since many of these aspects have been described widely for S. aureus, which is a frequent etiological agent of septic arthritis and osteomyelitis, this bacterium was included in parallel in most experiments for comparison.     

Administration of lysine acetylsalicylate and meperidine in acute postoperative pain]

INTRODUCTION. Postoperative analgesia is insufficiently done due, among others, to the undesirable effects of analgesic agents. Objective: The aim of this study was to analyze the effects of the simultaneous administration of opiates (meperidine) and AINES (lysine acetylsalicylate, ASL). MATERIAL AND METHODS. We studied 160 patients during the immediate postoperative phase. All of them underwent programmed surgery with the same general anesthetic technique. Patients were allocated into 8 groups of treatment: A) ASL 900 mg and 1.800 mg/8 h, B) ASL 900 mg and 3.600 mg/8 h, C) ASL 900 mg and meperidine 100 mg/8 h, D) ASL 900 mg and 1.800 mg/8 h together with meperidine 100 mg/8 h, E) meperidine 50 mg and ASL 1.800 mg/8 h, F) meperidine 50 mg and ASL 3.600 mg/8 h, G) meperidine 50 mg and 100 mg/8 h, and H) meperidine 50 mg and 100 mg/8 h together with ASL 1.800 mg/8 h. The effects of analgesic agents were evaluated on the basis of patient's appreciation of the degree of pain and relief and on the basis of an observer who did not know the therapeutic regime administered. Results were compared according to the analysis of variance in a graded factorial design. A p value less than 0.05 was considered significant. RESULTS. The degree of pain was significantly lower in groups C, D, G and H (specially in G and H) than in the remaining groups, but there were no significant differences between them. The lowest pain relief was observed in groups A, B, E and F. The highest attenuation of pain was achieved in groups G and H. The highest attenuation of pain was achieved in groups G and H. The observer considered that the two latter groups were those with the highest pain relief, followed by groups C and D. The remaining patients failed to show appreciable improvement. Nausea and vomiting only occurred in some patients after administration of a bolus of meperidine. There were no other secondary effects. CONCLUSIONS. The best degree of postoperative analgesia is achieved after administration of continuous infusion of meperidine 100 mg/8 h. Simultaneous infusion of ASL 1.800 mg/8 h did not improve the analgesia obtained with a bolus of 900 mg of ASL nor with a bolus of 50 mg of meperidine. Secondary effects were only nausea and vomiting and coincided with the administration of a bolus of meperidine.     

Acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas and other malignancies in patients with hemophilia

Non-Hodgkin's lymphoma (NHL) is the most common human immunodeficiency virus (HIV)-associated malignancy in hemophiliacs. We studied the incidence and clinicopathologic features of NHL in 3,041 hemophiliacs followed at 18 US Hemophilia Centers between 1978 and 1989. Of the 1,295 (56.6%) who were HIV(+), 253 (19.5%) developed acquired immunodeficiency syndrome (AIDS), of whom 14 (5.5%) developed NHL. Three NHL occurred in HIV(-) hemophiliacs, for a 36.5-fold greater risk in HIV(+) than HIV(-) hemophiliacs (P < .001). The NHL incidence rate was 29-fold greater than in the US population by Surveillance, Epidemiology, and End Results (SEER) estimates (P < .001). Between 0 and 4 lymphomas have been observed per year between 1978 and 1989. At presentation 13 (92.9%) of the HIV(+) NHL were extranodal. Ten were stage IV, 1 stage II, and 3 stage IE. Ten (71.4%) were high-grade, 3 (21.4%) intermediate-grade, and 1 (7.1%) was a low-grade B-cell lymphoma. Epstein-Barr virus (EBV) DNA was detected in 36% by in situ hybridization, including one central nervous system (CNS) lymphoma. The mean CD4 cell count at NHL diagnosis was 64/mm3, the mean latency from initial HIV infection was estimated to be 59 months, and the median survival was 7 months. The incidence of basal cell carcinoma in HIV(+) hemophiliacs was 18.3-fold greater than in HIV(-) hemophiliacs (P < .001) and 11.4-fold greater than in the US population (P < .001). In conclusion, incidence rates of NHL and basal cell carcinoma in HIV(+) hemophiliacs are significantly increased over rates in HIV(-) hemophiliacs and over rates in the US population. Clinicopathologic presentation of NHL in HIV(+) hemophiliacs is similar to that in HIV(+) homosexual men.