Improving antibiotic treatment of bacterial biofilm by hyperbaric oxygen therapy: Not just hot air.

Bacteria and fungi show substantial increased recalcitrance when growing as infectious biofilms. Chronic infections caused by biofilm growing microorganisms is considered a major problem of modern medicine. New strategies are needed to improve antibiotic treatment of biofilms. We have improved antibiotic treatment of bacterial biofilms by reviving the dormant bacteria and thereby make them susceptible to antibiotics by means of reoxygenation. Here we review the rationale for associating lack of oxygen with low susceptibility in infectious biofilm, and how hyperbaric oxygen therapy may result in reoxygenation leading to enhanced bactericidal activity of antibiotics. We address issues of feasibility and potential adverse effects regarding patient safety and development of resistance. Finally, we propose means for supplying reoxygenation to antibiotic treatment of infectious biofilm with the potential to benefit large groups of patients.

Hyperbaric oxygen therapy augments tobramycin efficacy in experimental Staphylococcus aureus endocarditis.

Staphylococcus aureus infective endocarditis (IE) is a serious disease with an in-hospital mortality of up to 40%. Improvements in the effects of antibiotics and host responses could potentially benefit outcomes. Hyperbaric oxygen therapy (HBOT) represents an adjunctive therapeutic option. In this study, the efficacy of HBOT in combination with tobramycin in S. aureus IE was evaluated. A rat model of S. aureus IE mimicking the bacterial load in humans was used. Infected rats treated subcutaneously with tobramycin were randomised into two groups: (i) HBOT twice daily (n = 13); or (ii) normobaric air breathing (non-HBOT) (n = 17). Quantitative bacteriology, cytokine expression, valve vegetation size and clinical status were assessed 4 days post-infection. Adjunctive HBOT reduced the bacterial load in the aortic valves, myocardium and spleen compared with the non-HBOT group (P = 0.004, <0.001 and 0.01, respectively) and improved the clinical score (P <0.0001). Photoplanimetric analysis and weight of valve vegetations showed significantly reduced vegetations in the HBOT group (P <0.001). Key pro-inflammatory cytokines [IL-1ß, IL-6, keratinocyte-derived chemokine (KC) and vascular endothelial growth factor (VEGF)] were significantly reduced in valves from the HBOT group compared with the non-HBOT group. In conclusion, HBOT augmented tobramycin efficacy as assessed by several parameters. These findings suggest the potential use of adjunctive therapy in severe S. aureus IE.

Incidence of Propionibacterium acnes in initially culture-negative thioglycollate broths-a prospective cohort study at a Danish University Hospital.

The aim of this study was to prospectively investigate the incidence of Propionibacterium acnes in thioglycollate broths reported as culture-negative at the Department of Clinical Microbiology, Rigshospitalet, to evaluate whether 5 days of incubation was enough to find all relevant cases. Five hundred thioglycollate broths reported as culture-negative after 5 days were consecutively collected and incubated for at least a further 9 days (at least 14 days of incubation in total). Only tissue samples from sterile sites of the body (n = 298), bone tissue (n = 197) and foreign material (n = 5) were included in this study. Samples were divided into two groups: infected group and control group. This made it possible to compare findings between groups, thereby making it possible to estimate the level of true-positive findings and contamination. Samples from 296 participants were included in this study. After exclusion criteria were met, P. acnes was cultured from ten out of 151 patients (6.6%) in the infected group and from one out of 138 participants (0.7%) in the control group. This resulted in more findings of P. acnes in the infected group on day 14 than on day 5 (p 0.002). Furthermore, P. acnes was cultured more often from bone tissue and tissue surrounding foreign materials on day 14 than on day 5 (p 0.04). Clinical microbiology laboratories should consider incubating thioglycollate broths for at least 14 days to find all relevant cases of P. acnes, especially when it comes to bone tissue and tissue surrounding foreign materials.

Anti-Pseudomonas aeruginosa IgY antibodies augment bacterial clearance in a murine pneumonia model.

BACKGROUND: Oral prophylactic therapy by gargling with pathogen-specific egg yolk immunoglobulins (IgY) may reduce the initial airway colonization with Pseudomonas aeruginosa in cystic fibrosis (CF) patients. IgY antibodies impart passive immunization and we investigated the effects of anti-P. aeruginosa IgY antibodies on bacterial eradication in a murine pneumonia model. METHODS: P. aeruginosa pneumonia was established in Balb/c mice and the effects of prophylactic IgY administration on lung bacteriology, clinical parameters and subsequent inflammation were compared to controls. RESULTS: Prophylactic administration of IgY antibodies targeting P. aeruginosa significantly reduced the bacterial burden by 2-log 24h post-infection compared to controls and was accompanied by significantly reduced clinical symptom scores and successive inflammatory cytokine profile indicative of diminished lung inflammation. CONCLUSIONS: Passive immunization by anti-P. aeruginosa IgY therapy facilitates promptly bacterial clearance and moderates inflammation in P. aeruginosa lung infection and may serve as an adjunct to antibiotics in reducing early colonization.

Low efficacy of tobramycin in experimental Staphylococcus aureus endocarditis.

The empiric treatment of infective endocarditis (IE) varies widely and, in some places, a regimen of penicillin in combination with an aminoglycoside is administered. The increasing incidence of Staphylococcus aureus IE, poor tissue penetration by aminoglycosides and low frequency of penicillin-susceptible S. aureus may potentially lead to functional tobramycin monotherapy. Therefore, this study aimed to evaluate tobramycin monotherapy in an experimental S. aureus IE rat model. Catheter-induced IE at the aortic valves were established with S. aureus (NCTC 8325-4) and rats were randomised into untreated (n = 22) or tobramycin-treated (n = 13) groups. The treatment group received tobramycin once-daily. Animals were evaluated at 1 day post infection (DPI), 2 DPI or 3 DPI. Quantitative bacteriology and cytokine expression were measured for valves, myocardium and serum. A decrease of bacterial load was observed in valves and the spleens of the treated (n = 6) compared to the untreated group at 2 DPI (n = 8) (p ≤ 0.02 and p ≤ 0.01, respectively), but not at 3 DPI (n = 7). Quantitative bacteriology in the myocardium was not different between the groups. Keratinocyte-derived chemokine (KC) in the aortic valves was significantly reduced at 2 DPI in the tobramycin-treated group (p ≤ 0.03). However, the expression of interleukin (IL)-1b, IL-6 and granulocyte-colony stimulating factor (G-CSF) in the valves was not different between the two groups. In the myocardium, a significant reduction in IL-1b was observed at 2 DPI (p ≤ 0.001) but not at 3 DPI. Tobramycin as functional monotherapy only reduced bacterial load and inflammation transiently, and was insufficient in most cases of S. aureus IE.

Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils.

Polymorphonuclear neutrophils (PMNs) are essential cellular constituents in the innate host response, and their recruitment to the lungs and subsequent ubiquitous phagocytosis controls primary respiratory infection. Cystic fibrosis pulmonary disease is characterized by progressive pulmonary decline governed by a persistent, exaggerated inflammatory response dominated by PMNs. The principal contributor is chronic Pseudomonas aeruginosa biofilm infection, which attracts and activates PMNs and thereby is responsible for the continuing inflammation. Strategies to prevent initial airway colonization with P. aeruginosa by augmenting the phagocytic competence of PMNs may postpone the deteriorating chronic biofilm infection. Anti-P. aeruginosa IgY antibodies significantly increase the PMN-mediated respiratory burst and subsequent bacterial killing of P. aeruginosa in vitro. The mode of action is attributed to IgY-facilitated formation of immobilized bacteria in aggregates, as visualized by fluorescence microscopy and the induction of increased bacterial hydrophobicity. Thus, the present study demonstrates that avian egg yolk immunoglobulins (IgY) targeting P. aeruginosa modify bacterial fitness, which enhances bacterial killing by PMN-mediated phagocytosis and thereby may facilitate a rapid bacterial clearance in airways of people with cystic fibrosis.

Nitric oxide production by polymorphonuclear leucocytes in infected cystic fibrosis sputum consumes oxygen.

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O(2)) due to production of superoxide (O(2)(-)). In this study, we show that the PMNs also consume O(2) for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with N(G) -monomethyl-L-arginine (L-NMMA) resulted in reduced O(2) consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO(3)(-) (P < 0·04, r = 0·66, n = 10) and NO(2)(-) (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O(2) for production of reactive oxygen species, the PMNs in CF sputum also consume O(2) for production of NO.

Bead-size directed distribution of Pseudomonas aeruginosa results in distinct inflammatory response in a mouse model of chronic lung infection.

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by biofilms, tolerant to antibiotics and host responses. Instead, immune responses contribute to the tissue damage. However, this may depend on localization of infection in the upper conductive or in the peripheral respiratory zone. To study this we produced two distinct sizes of small alginate beads (SB) and large beads (LB) containing P. aeruginosa. In total, 175 BALB/c mice were infected with either SB or LB. At day 1 the quantitative bacteriology was higher in the SB group compared to the LB group (P < 0·003). For all time-points smaller biofilms were identified by Alcian blue staining in the SB group (P < 0·003). Similarly, the area of the airways in which biofilms were identified were smaller (P < 0·0001). A shift from exclusively endobronchial to both parenchymal and endobronchial localization of inflammation from day 1 to days 2/3 (P < 0·05), as well as a faster resolution of inflammation at days 5/6, was observed in the SB group (P < 0·03). Finally, both the polymorphonuclear neutrophil leucocyte (PMN) mobilizer granulocyte colony-stimulating factor (G-CSF) and chemoattractant macrophage inflammatory protein-2 (MIP-2) were increased at day 1 in the SB group (P < 0·0001). In conclusion, we have established a model enabling studies of host responses in different pulmonary zones. An effective recognition of and a more pronounced host response to infection in the peripheral zones, indicating that increased lung damage was demonstrated. Therefore, treatment of the chronic P. aeruginosa lung infection should be directed primarily at the peripheral lung zone by combined intravenous and inhalation antibiotic treatment.

Genetical analysis of all Danish patients diagnosed with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disorder of the innate immune system caused by a defect in NADPH oxidase, leaving the granulocytes unable to kill invading microorganisms. CGD is caused by mutation in one of the five components gp91phox, p22phox, p47phox, p67phox and p40phox, encoded by the X-linked CYBB gene and the autosomal CYBA, NCF1, NCF2 and NCF4 genes respectively. We have collected samples from all Danish patients with known CGD followed in the clinic or newly diagnosed during a 5-year period, a cohort of 27 patients, and characterized them genetically. The cohort includes 10 male patients with X-linked CGD and one female with extremely lyonized expression of a defective CYBB allele. Six patients had mutation in CYBA. Seven of 10 patients with a defect in NCF1 were homozygous for the common GT deletion, one was compound heterozygous for the GT deletion and a splice-site mutation, and two patients were homozygous for a nonsense mutation in exon 7. Three novel mutations were detected, a deletion of exon 6 in CYBA, a duplication of exon 8-13 in CYBB and a splice site mutation in intron 7 of NCF1.

Inflammation in Achromobacter xylosoxidans infected cystic fibrosis patients.

BACKGROUND: Achromobacter xylosoxidans infection may cause conspicuous chronic pulmonary inflammation in cystic fibrosis (CF) patients similar to Pseudomonas aeruginosa and the Burkholderia cepacia complex (Bcc). Evolution in lung function was compared in chronically infected patients. Cytokine concentrations in CF patients with and without chronic infection were compared to healthy controls. METHODS: Cytokines in serum and sputum were measured using multiplex bead based immunoassay. RESULTS: Sixty CF patients, 11 with A. xylosoxidans, 11 with Bcc, 21 with P. aeruginosa and 17 non-infected CF patients were compared to 11 healthy controls. A. xylosoxidans patients were younger, but had a FEV(1) decline similar to P. aeruginosa patients. Bcc patients had the steepest decline in FEV(1). Serum levels of G-CSF, IL-6 and TNF-alpha were significantly higher in CF patients compared to healthy controls. Chronically infected CF patients had significantly higher serum levels of IFN-gamma and IL-6 compared to non-infected CF patients. Bcc patients had significantly lower serum G-CSF and A. xylosoxidans patients had significantly higher sputum TNF-alpha compared to the other groups of chronically infected patients. CONCLUSION: A. xylosoxidans can cause a level of inflammation similar to P. aeruginosa in chronically infected CF patients. A. xylosoxidans is a clinically important pathogen in CF and should be treated accordingly.

Polymorphonuclear leucocytes consume oxygen in sputum from chronic Pseudomonas aeruginosa pneumonia in cystic fibrosis.

BACKGROUND: Chronic lung infection with Pseudomonas aeruginosa is the most severe complication for patients with cystic fibrosis (CF). This infection is characterised by endobronchial mucoid biofilms surrounded by numerous polymorphonuclear leucocytes (PMNs). The mucoid phenotype offers protection against the PMNs, which are in general assumed to mount an active respiratory burst leading to lung tissue deterioration. An ongoing respiratory burst by the PMNs has, however, not been demonstrated previously in endobronchial secretions from chronically infected patients with CF. OBJECTIVE: Based on the accumulating evidence for depletion of molecular oxygen (O(2)) in the mucus in infected CF bronchi, it was hypothesised that the O(2) depletion in the mucus in infected CF bronchi may be accelerated by the respiratory burst of the PMNs due to the reduction of O(2) to the superoxide anion (O(-)(2)) by the phagocyte NADPH oxidase (Phox). METHODS: Methods were established to isolate the O(2) consumption by the respiratory burst from aerobic respiration in freshly expectorated sputum from chronically infected patients with CF. RESULTS: Inhibition of the Phox with diphenylene iodonium (DPI) delayed O(2) depletion, nearly abolished staining of O(-)(2)-producing PMNs with hydroethidine and inhibited the rapid luminol-enhanced chemiluminescence in sputum. Furthermore, the total O(2) consumption was correlated to the concentration of PMNs in the sputum samples. CONCLUSION: The results demonstrate that CF sputum contains PMNs with an active consumption of O(2) for O(-)(2) production and suggest that the respiratory burst is ongoing and causes accelerated O(2) depletion due to formation of O(-)(2) in the lungs of chronically infected patients with CF.

Thermal injury induces impaired function in polymorphonuclear neutrophil granulocytes and reduced control of burn wound infection.

Severe thermal injury induces immunosuppression, involving all parts of the immune system, especially when large fractions of the total body surface area are affected. An animal model was established to characterize the burn-induced immunosuppression. In our novel mouse model a 6% third-degree burn injury was induced in mice with a hot-air blower. The third-degree burn was confirmed histologically. The mice were allocated into five groups: control, shave, burn, infection and burn infection group. At 48 h, a decline in the concentration of peripheral blood leucocytes was observed in the group of mice with burn wound. The reduction was ascribed to the decline in concentration of polymorphonuclear neutrophil leucocytes and monocytes. When infecting the skin with Pseudomonas aeruginosa, a dissemination of bacteria was observed only in the burn wound group. Histological characterization of the skin showed a more polymorphonuclear neutrophil granulocytes (PMNs)-dominated inflammation in the group of mice with infected burn wound compared with the with burn wound group. In contrast, a higher degree of inflammation was observed in the burn wound group compared with the group of mice with infected burn wound. Furthermore, the oxidative burst and the phagocytic capacity of the PMNs were reduced in the group of mice with burn wound. Using this novel mouse model of thermal injury a decline of peripheral leucocytes was observed, whereas the increased local inflammatory response at the site of infection showed reduced capacity to contain and eliminate the infection.

Increased serum concentration of G-CSF in cystic fibrosis patients with chronic Pseudomonas aeruginosa pneumonia.

BACKGROUND: Chronic Pseudomonas aeruginosa lung infection is the major reason for premature death in patients with cystic fibrosis (CF). Infected patients experience a progressive deterioration of the lung tissue caused by a persistent accumulation of PMNs. We investigated if the pulmonary accumulation of PMNs is reflected as a migration of PMNs through the blood in chronically infected CF patients. METHODS: Blood and sputum samples from 37 stable, chronically (CF+P) and 6 non-infected (CF-P) CF patients without exacerbations were compared using FACS, leukocyte counting, and ELISA. Within the CF+P patients, the blood parameters were compared to the lung function (FEV1 and FVC) and to the sputum. Similar measurements were performed on 15 chronically infected CF patients before and after elective antibiotic treatment. RESULTS: In the CF+P patients the concentration of G-CSF in the sera and PMNs in the blood was increased and correlated to poor lung function. However, only the concentration of G-CSF in the sera was correlated to the concentration of TNF-alpha in the sputum. After the antibiotic treatment, the lung function was improved and the concentration of PMNs in the blood and G-CSF in the sera was reduced. CONCLUSION: G-CSF in the sera may contribute to the pulmonary inflammation in CF patients with chronic P. aeruginosa lung infection by regulating the number of PMNs available for migration and may be considered as an indicator of clinical status.

Faster activation of polymorphonuclear neutrophils in resistant mice during early innate response to Pseudomonas aeruginosa lung infection.

Polymorphonuclear neutrophils (PMNs) are crucial for the outcome of Pseudomonas aeruginosa lung infection in patients with cystic fibrosis. We compared PMNs and inflammatory cytokines in the lungs and blood from susceptible BALB/c and resistant C3H/HeN mice 1 and 2 days after intratracheal challenge with alginate embedded P. aeruginosa. These parameters were correlated with the quantitative bacteriology and histopathology of the lungs. After challenge, the content of granulocyte colony-stimulating factor (G-CSF) and macrophage inflammatory protein-2 (MIP-2) was increased in the lungs and the sera and the percentage of PMNs was increased in the blood. However, 2 days after challenge the concentration of G-CSF and MIP-2 was higher in the lungs and sera of BALB/c mice. CD11b expression was higher on the PMNs of the C3H/HeN mice. The expression of CD62L on PMNs of both strains of mice was decreased 1 day after bacterial challenge, whereas the expression was increased after 2 days of challenge on PMNs of C3H/HeN mice only. These changes were accompanied by a more severe lung inflammation in BALB/c mice and faster clearance of the bacteria in C3H/HeN mice. In conclusion, the rapid early bacterial clearance in the lungs of C3H/HeN mice could be explained by faster activation of the PMNs, as indicated by the higher up-regulation of CD11b. The severe lung inflammation in BALB/c mice may be caused by the early higher content of G-CSF in the sera mobilizing PMNs from the bone marrow and the persistent chemotactic gradient provided by MIP-2 in the lungs.

Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response.

Repeated challenge with antigen is involved in the pathogenesis of a variety of pulmonary diseases. Patients with cystic fibrosis (CF) experience recurrent pulmonary colonization with Pseudomonas aeruginosa before establishment of chronic lung infection. To mimic recurrent lung infections in CF patients, the lungs of susceptible BALB/c mice were re-infected with P. aeruginosa 14 days after the initial infection. Singly-infected BALB/c mice, as well as non-infected mice, were used as controls. Decreased mortality and milder lung inflammation in re-infected BALB/c mice, as well as a tendency for improved clearance of bacteria, was observed when compared with singly-infected mice. The improved outcome in re-infected mice correlated with changes in CD4 cell numbers. Surface expression of LFA-1 on pulmonary CD4 cells was increased in re-infected compared with singly-infected mice. Moreover, resistance to re-infection was paralleled by a shift towards a Th1-dominated response and increased IL-12 production. No significant increase in serum IgG was observed in the re-infected mice. In conclusion, these results indicate a protective role for a Th1-dominated response, independent of antibody production, in chronic P. aeruginosa lung infection in CF.

Flow cytometric analysis of RNA synthesis by detection of bromouridine incorporation.

RNA synthesis has traditionally been investigated by a laborious and time-consuming radiographic method involving incorporation of tritiated uridine. Now a faster non-radioactive alternative has emerged, based on immunocytochemical detection. This method utilizes the brominated RNA precursor bromouridine, which is taken into a cell, phosphorylated, and incorporated into nascent RNA. The BrU-substituted RNA is detected by permeabilizing the cells and staining with certain anti-BrdU antibodies. This dynamic approach yields information complementing that provided by cellular RNA content analysis at a given time and may be of value in studies of cellular activation and gene expression.

Increased cellular hypoxia and reduced proliferation of both normal and leukaemic cells during progression of acute myeloid leukaemia in rats.

The microenvironmental changes in the bone marrow, spleen and liver during progression of the transplantable promyelocytic leukaemia in the Brown Norwegian rat (BNML) have been studied. We used flow cytometry to estimate cellular hypoxia and proliferation based on in vivo pulse-labelling with a mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27 in the bone marrow and liver, reaching a level of 65-87% in these organs at day 32. At day 32, the NITP+ fraction of RM124+ cells had increased significantly in the bone marrow and spleen to 88% and 90%, respectively. The corresponding fractions of NITP+ normal cells reached 63% and 65%, respectively. From day 13 to day 32, the DNA-synthesizing (BrdUrd+) fraction of RM124+ cells in the bone marrow decreased significantly from 52% to 25%, and of normal cells from about 20% to 6%. In the bone marrow and spleen at day 27 and 32, the S-phase and G2/M-phase fractions according to DNA content were higher for the NITP+ than for the NITP- cells. This could partly be explained by an impaired cell cycle progression due to hypoxia. Nevertheless, we found indications of leukaemic cells that were simultaneously labelled with NITP and BrdUrd, in the bone marrow and spleen. These latter findings suggest that in contrast to normal cells some of the leukaemic cells can proliferate even during hypoxia, and this subpopulation may consequently renew and expand the leukaemic cell load.

S-phase induction by interleukin-6 followed by chemotherapy in patients with chronic lymphocytic leukemia and non-Hodgkin's lymphoma.

Interleukin-6 (IL-6) has in vitro demonstrated growth regulatory effects on tumor cells from patients with chronic lymphocytic leukemia (CLL) and lymphoma. The proliferation rate of these cells is usually very low and this is thought to be one of the reasons for the lack of a curative potential of cytostatic chemotherapy in CLL and low grade NHL. Recombinant human (rh) IL-6 might increase the in vivo proliferation rate leading to a higher sensitivity for chemotherapy. We tested this hypothesis by administering rhIL-6 to 9 CLL patients and 3 NHL patients in doses of 2.5 micrograms/kg, 5 micrograms/kg and 10 micrograms/kg s.c. daily for 5 days followed by CHOP chemotherapy on the last day of rhIL-6 injection. Six patients had two treatment cycles. The proportion of cells in S-phase was determined by the bromodeoxyuridine labeling index (LI). Three patients achieved a partial remission, one patient had progressive disease and the remaining patients demonstrated no change. Two patients, who received 10 micrograms/kg/day rhIL-6, demonstrated a significant increase in LI, one of these was first observed in the second treatment cycle. A significant decrease was seen in two patients receiving 2.5 micrograms/kg and 5 micrograms/kg respectively. Immunophenotypic assessment demonstrated that rhIL-6 increased the expression of CD20 in all CLL patients with a reversal after cessation of rhIL-6. We conclude that rhIL-6, in the dosage and schedule used in this study, did not increase the proportion of the cells in S-phase and that the growth stimulatory effects of rhIL-6 in CLL in vivo probably are insignificant. However, the role of rhIL-6 in CLL as inducer of increased CD20 expression prior to anti-CD20 antibody treatment remains to be determined.

S-phase induction by interleukin-6 followed by chemotherapy in patients with refractory multiple myeloma.

The plasma cell labeling index (PCLI) in patients with multiple myeloma (MM) is relatively low and this has been associated with the low rate of remission following chemotherapy. Interleukin-6 (IL-6) has been demonstrated to be a major growth factor of myeloma cells. In order to increase the S-phase proportion of myeloma cells, which might increase the sensitivity to chemotherapy, we gave rhIL-6 followed by chemotherapy to 15 myeloma patients with refractory disease. A total of 25 treatment cycles were administered since ten patients had two cycles. The rhIL-6 dose was 2.5 (n = 3), 5.0 (n = 6) and 10.0 microg/kg (n = 6) by subcutaneous injection once daily for 5 days and chemotherapy was administered on the last day of rhIL-6 injection. The effect of rhIL-6 treatment on labeling index (LI) was heterogeneous, but no statistically significant change was noted for this particular group as a whole. In two patients an increase (mean 7.7%) in LI of mononuclear bone marrow cells during the rhIL-6 treatment was demonstrated and in one patient a decrease of 2.8% was seen. Assessment of PCLI demonstrated an increase of 2.9% in one out of six patients and a decrease of 1.9% in one out of six patients. None of the 15 patients achieved remission according to standard criteria. During the rhIL-6 treatment, 14 of the 15 patients developed mild constitutional adverse events (AE) well known in patients treated with IL-6, and none of the AE in the subsequent chemotherapy phase were related to IL-6. In conclusion, our study demonstrated that rhIL-6 can be administered safely to patients with refractory MM, but the cell cycle recruitment approach was not sufficiently effective to be of clinical value.