R-CHOEP-14 improves overall survival in young high-risk patients with diffuse large B-cell lymphoma compared with R-CHOP-14. A population-based investigation from the Danish Lymphoma Group.

BACKGROUND: Optimal treatment of young patients with high-risk diffuse large B-cell lymphoma (DLBCL) remains a matter of debate and requires improvement. The combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) with addition of etoposide (CHOEP) has in other patient groups been shown to be effective. Further improvement has been accomplished with the use of rituximab in combination with the regimens every 2 weeks (R-CHOP-14, R-CHOEP-14). The aim of the present retrospective population-based study was to compare R-CHOP-14 with R-CHOEP-14 in a cohort of high-risk patients aged 18-60 years with two or more risk factors (stage III-IV, elevated lactate dehydrogenase levels, performance status 2-4). To our knowledge, this is the first study comparing these two regimens in this patient group. METHODS: We obtained data for the period 2004-2009 from the Danish Lymphoma Database. One hundred and fifty-nine patients were eligible to enter the study. Primary end point was overall survival (OS) and secondary end points were response to treatment, progression-free survival (PFS) and safety. RESULTS: Four-year OS was superior in the R-CHOEP-14 group: 75% compared with 62% for R-CHOP-14 (P=0.04). This superiority was also seen for PFS: 4-year PFS was 70% for the R-CHOEP-14 group compared with 58% for the R-CHOP-14 group (P=0.02). CONCLUSION: R-CHOEP-14 is a promising regimen for young patients with high-risk DLBCL with improved OS and PFS compared with R-CHOP-14.

[Neonatal conjunctivitis after the abolition of compulsory Credé prophylaxis]. / Conjunctivitis neonatalis efter ophør med obligatorisk Credé-profylakse.

The prescript requiring application of silver nitrate eye drops (0.66% AgNO3) to the conjunctivae of the newborn within two hours after delivery was revoked in March 1985. The present study comprises a prospective investigation of the occurrence of microorganisms in specimens of eye secretion from neonates received during the period February-April 1986, and a review of findings of Neisseria species and B. catarrhalis in 3,485 specimens of ocular secretions and of C. trachomatis in 1,240 conjunctival scrapes received at the Neisseria Department 1986-1988. The numbers of cases of conjunctivitis neonatalis caused by the following microorganisms were: N. gonorrhoeae 8, N. cinerea 6, other Neisseria species 8, B. catarrhalis 49 and C. trachomatis 92. During the period 1984-1988, the total number of neonates with gonococcal conjunctivitis neonatalis was 18. The gonococcal infection was diagnosed within the first week of life in 50% of the cases but could occur as late as in the fourth week of life. Similarly, infection with B. catarrhalis was most common in neonates less than one week old (49%), whereas chlamydial infection was most common in the second week of life (39%). It is concluded that the eyes of neonates should be carefully observed for at least four weeks and that microbiological examinations for gonococci and chlamydia are indicated if signs of infection appear.

In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes. Inhibition by alpha-hemolysin antibody.

The influence of alpha-hemolysin antibody on the in vitro cytotoxic effect of alpha-hemolytic Escherichia coli bacteria and culture filtrates was investigated. Damage to human blood granulocytes was quantified by measuring the release of chromium 51 from labelled cells in the presence of whole or fractionated plasma containing alpha-hemolysin antibody. Anti-alpha-hemolysin activity was found exclusively in the IgG fraction of plasma. Human plasma contained "natural" alpha-hemolysin antibody to various titers. Vaccination of rabbits resulted in only modest rises in alpha-hemolysin antibody titers. The cytotoxic effect of alpha-hemolytic E. coli bacteria as well as that of bacterial culture filtrates was reduced in a titer-dependent way in the presence of plasma containing alpha-hemolysin antibody. These results indicate that the cytotoxic effect of alpha-hemolytic E. coli is inhibited by alpha-hemolysin antibody.

Role of alpha-hemolysin for the in vitro phagocytosis and intracellular killing of Escherichia coli.

The role of alpha-hemolysin for the elimination of Escherichia coli by phagocytes in vitro was investigated using sets of isogenic strains which included wild-type alpha-hemolytic strains, derived strains with a reduced production of alpha-hemolysin and derived nonhemolytic strains. Phagocytosis and intracellular killing of the bacteria by human blood granulocytes or monocytes were measured using growth inhibition techniques. alpha-hemolytic strains were phagocytosed and killed to a lesser extent than isogenic strains with a reduced production of alpha-hemolysin and isogenic nonhemolytic strains. The results obtained with granulocytes were similar to those obtained with monocytes although the elimination of bacteria by monocytes was less than that by granulocytes. These results strongly suggest that production of alpha-hemolysin is a means by which E. coli counteracts the activity of phagocytes by injuring these cells with the toxin.

In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes. Correlation with size of alpha-hemolysin production.

The correlation of the in vitro cytotoxic effect of 107 alpha-hemolytic strains of Escherichia coli with various other bacterial characteristics was investigated. Damage to human blood granulocytes in the presence of fresh or heated autologous plasma was quantified by measuring the release of chromium-51 from labelled cells. 95 strains had a cytotoxic effect which was equal in the presence of fresh or heated plasma, whereas 12 strains showed an effect which was reduced in fresh compared with heated plasma. The cytotoxic effect increased as the number of bacteria per granulocyte was increased. The average size of the alpha-hemolysin production of the strains was 185 HU50/ml ranging from 3-2519HU50/ml. The cytotoxic effect of the strains was directly correlated with the size of the alpha-hemolysin production. The cytotoxic effect was not correlated with the O-antigen serotype or the type of infection from which the strains were derived. These results indicate that the ability to produce alpha-hemolysin is the bacterial characteristic which is of decisive importance for the cytotoxicity of alpha-hemolytic E. coli towards human blood granulocytes.

In vitro cytotoxic effect of alpha-hemolytic and nonhemolytic Escherichia coli on human blood granulocytes and monocytes.

The in vitro cytotoxic effect of Escherichia coli on human phagocytic blood cells in the presence of fresh autologous plasma was assessed using (i) a technique by which cell damage was quantified by measuring the release of chromium 51 from labelled granulocytes and (ii) a technique based on the degree of morphological cell damage induced in monocytes. All of 109 alpha-hemolytic strains were cytotoxic, the cytotoxic effect ranging from very weak to strong. Log-phase cultures had the strongest cytotoxic effect, the individual microorganisms being most cytotoxic in the early log-phase. Plateau-phase cultures progressively lost their cytotoxic ability and 24 h old cultures were almost noncytotoxic. Strongly cytotoxic strains induced high degrees of cell injury, caused an abrupt damage during the first 20 min of incubation and were able to induce injury with few bacteria present. Weakly cytotoxic strains induced only low degrees of cell injury, caused a more gradually developing damage, and were able to induce injury with only high numbers of bacteria present. All of 75 nonhemolytic and 3 beta-hemolytic strains were noncytotoxic.

Morphological study of the in vitro cytotoxic effect of alpha-hemolytic E. coli bacteria and culture supernatants on human blood granulocytes and monocytes.

The morphological changes of human blood granulocytes and monocytes caused in vitro by alpha-hemolytic strains of E. coli and bacteria-free culture supernatants of these bacteria were studied by light- and transmission electron microscopy. The following sequence of cellular alterations were observed: Cessation of intracellular cytoplasmic streaming and cellular movements succeeded by extension of cytoplasmic pseudopodia, degranulation and development of cytoplasmic and nuclear edema. Within two hours the leukocytes appeared as empty sacks. Finally, long straight filaments were formed between the cells. The changes induced by alpha-hemolytic bacteria and culture supernatants containing free alpha-hemolysin appeared to be identical. The cytotoxic effect became more pronounced as the numbers of bacteria, the hemolytic activity of growth supernatants or the period of incubation were increased. A beta-hemolytic and a nonhemolytic E. coli strain were not cytotoxic.

In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes.

The cytotoxic effect of Escherichia coli bacteria on human blood granulocytes was measured by recording numbers of nonlysed cells and percentages of viable cells after in vitro incubation with bacteria in the presence of plasma. A total of 179 strains from various sources of infection were tested. Of 117 alpha-hemolytic strains, 59 were cytotoxic. Five nonhemolytic mutant strains, derived from alpha-hemolytic cytotoxic strains, were nontoxic. None of the 62 nonhemolytic strains were toxic. Four spontaneously occurring alpha-hemolytic, nontoxic mutant strains were isolated from cytotoxic ones. Cytotoxicity of bacteria reached a maximum after log-phase growth at 30 to 37 degrees C for 2.5 h, and the toxic capacity was equal after growth in various media, including human urine and plasma. The cytotoxic effect increased with the length of exposure of granulocytes to bacteria and with increasing numbers of bacteria per granulocyte. Cytotoxic strains showed different degrees of toxicity, highly cytotoxic strains lysing about 90% of the granulocytes and killing about one-half of nonlysed cells in 1 h. Bacteria killed by heat, formaldehyde, or UV light were nontoxic. Alpha-hemolytic strains of O groups 2, 4, 6, 25, and 75 originating from various infections in humans were more frequently cytotoxic than alpha-hemolytic strains of other O groups derived from human infections. Culture supernatants containing free alpha-hemolysin were highly cytotoxic to human blood granulocytes, monocytes, and lymphocytes in vitro, whether supernatants originated from cytotoxic or noncytotoxic bacteria. Cytotoxicity to phagocytes, which is mediated by or closely linked genetically to alpha-hemolysin, may be a mechanism by which alpha-hemolytic strains of E. coli strengthen their ability to establish and maintain infections.

Cytotoxic effect of an alpha-hemolytic Escherichia coli strain on human blood monocytes and granulocytes in vitro.

The alpha-hemolytic Escherichia coli strain C134-73 Hly+ was primarily cytocidal to human blood monocytes and granulocytes in vitro in the presence of fresh autologous plasma. Monocytes and granulocytes underwent marked morphological changes during incubation with the bacteria, and the percentages of intact phagocytes decreased progressively with the time of incubation. The cytotoxic effect increased with the number of bacteria per phagocyte and was produced by log-phase microorganisms only. Neither free hemolysin nor free cytotoxic activity to leukocytes could be detected in the incubation medium if the bacteria were removed from the test system. Bacteria-free culture supernatants containing alpha-hemolysin were cytotoxic to monocytes, granulocytes, and lymphocytes, monocytes and granulocytes being the most sensitive. However, this effect was abolished by fresh autologous plasma. Two nonhemolytic strains, a mutant derivative of C134-73 Hly+ and strain X43, were not cytotoxic. These observations suggest that alpha-hemolysin associated with the bacterial cells may enhance the virulence of E. coli by injuring phagocytes, whereas free alpha-hemolysin may be of minor importance because its cytotoxic effect is neutralized by host plasma.

Comparison of smear and cytocentrifugation techniques for estimation of human peripheral blood monocytes.

Differential counts of smears and cytospins of full blood, dextran fractionated blood leukocytes and metrizoate/Ficoll fractionated mononuclear blood cells were compared. Frequencies of lymphocytes decreased in cytospins as compared with smears. The monocyte/lymphocyte ratios were 0.24 in smears of full blood and 0.22, 0.31, 0.15 and 0.39 in smears and cytospins of dextran supernatant cells and metrizoate/Ficoll interface cells, respectively. Less than 5% of cells were broken in all preparations. The slide to slide variation was larger in preparations of fractionated cells than in smears of full blood. Cells were evenly distributed in mononuclear cytospins.

The effect of various immunosuppressive agents on mouse peritoneal macrophages and on the in vitro phagocytosis of Escherichia coli O4:K3:H5 and degradation of 125I-labelled HSA-antibody complexes by these cells.

Large doses of hydrocortisone, cyclophosphamide, and methotrexate injected subcutaneously, and whole-body irradiation (500 rads) caused a reduction in the number of peritoneal cells (PE cells) obtained after intraperitoneal injection of the treated mice with proteose-peptone. The same dose of cyclophosphamide and irradiation induced morphological changes in PE macrophages. There were more giant cells in the peritoneal exudates from treated mice as compared to control mice. 'Pharmacological' and larger doses of hydrocortisone, methotrexate and azathioprine or anti-lymphocyte globulin had no effect on the in vitro phagocytic capacity of proteose-peptone-stimulated mouse PE macrophages. This also applied to doses of up to 50 mg/kg of cyclophosphamide. In contrast, whole-body irradiation (500 rad) and 100 mg/kg of cyclophosphamide decreased the phagocytic capacity of mouse macrophages in vitro and reduced the ability of PE cells to degrade 125I-labelled HSA-antibody complexes in vitro. The greatest effect was noted 4-5 days after whole-body irradiation or four to five subcutaneous injections of cyclophosphamide.