Biofilm formation - what we can learn from recent developments.

Although biofilms have been observed early in the history of microbial research, their impact has only recently been fully recognized. Biofilm infections, which contribute to up to 80% of human microbial infections, are associated with common human disorders, such as diabetes mellitus and poor dental hygiene, but also with medical implants. The associated chronic infections such as wound infections, dental caries and periodontitis significantly enhance morbidity, affect quality of life and can aid development of follow-up diseases such as cancer. Biofilm infections remain challenging to treat and antibiotic monotherapy is often insufficient, although some rediscovered traditional compounds have shown surprising efficiency. Innovative anti-biofilm strategies include application of anti-biofilm small molecules, intrinsic or external stimulation of production of reactive molecules, utilization of materials with antimicrobial properties and dispersion of biofilms by digestion of the extracellular matrix, also in combination with physical biofilm breakdown. Although basic principles of biofilm formation have been deciphered, the molecular understanding of the formation and structural organization of various types of biofilms has just begun to emerge. Basic studies of biofilm physiology have also resulted in an unexpected discovery of cyclic dinucleotide second messengers that are involved in interkingdom crosstalk via specific mammalian receptors. These findings even open up new venues for exploring novel anti-biofilm strategies.

Patients unaware of their HIV infection until AIDS diagnosis in Sweden 1996-2002--a remaining problem in the highly active antiretroviral therapy era.

Our objective was to analyse the characteristics of patients who were unaware of their HIV infection until they developed AIDS, in the period after introduction of highly active antiretroviral therapy. The complete national register of HIV and AIDS cases reported to the Department of Epidemiology at the Swedish Institute for Infectious Disease Control 1996-2002 was searched for cases diagnosed with HIV less than three months before AIDS diagnosis (so-called "late testers"). Of a total of 487 patients with AIDS, reported during the seven-year period, 219 (45%) were late testers. Their proportion of all AIDS cases increased from 22% in 1996 to 58% in 2002. Heterosexual route of transmission, age greater than 40 years, and foreign origin were all significant risk factors for being a late tester. Intravenous drug users were associated with a highly significant reduced risk. The group without previously known HIV infection represents an increasing part of all cases of AIDS. From a disease control and from a medical perspective, it is important to study this group further and discover what measures are needed for earlier identification and access to medical care.

Cyclodextrin-catalyzed extraction of fluorescent sterols from monolayer membranes and small unilamellar vesicles.

This study examined the kinetics of sterol desorption from monolayer and small unilamellar vesicle membranes to 2-hydroxypropyl-beta-cyclodextrin. The sterols used include cholesterol, dehydroergosterol (ergosta-5,7,9,(11),22-tetraen-3beta-ol) and cholestatrienol (cholesta-5,7,9,(11)-trien-3beta-ol). Desorption rates of dehydroergosterol and cholestatrienol from pure sterol monolayers were faster (3.3-4.6-fold) than the rate measured for cholesterol. In mixed monolayers (sterol: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 30:70 mol%), both dehydroergosterol and cholestatrienol desorbed faster than cholesterol. clearly indicating a difference in interfacial behavior of these sterols. In vesicle membranes desorption of dehydroergosterol was slower than desorption of cholestatrienol, and both rates were markedly affected by the phospholipid composition. Desorption of sterols was slower from sphingomyelin as compared to phosphatidylcholine vesicles. Desorption of fluorescent sterols was also faster from vesicles prepared by ethanol-injection as compared to extruded vesicles. The results of this study suggest that dehydroergosterol and cholestatrienol differ from cholesterol in their membrane behavior, therefore care should be exercised when experimental data derived with these probes are interpreted.

N-acetylcysteine treatment and the risk of toxic reactions to trimethoprim-sulphamethoxazole in primary Pneumocystis carinii prophylaxis in HIV-infected patients.

In a randomized double blind placebo controlled trial, HIV sero-positive patients with CD4+ cell count less than 200 x 10(6)/l or an AIDS diagnosis were evaluated for drug reactions to trimethoprim-sulphamethoxazole (TMP-SMX) during treatment, including pretreatment, with N-acetylcysteine (NAC) 800 mg daily or placebo. TMP-SMX (one double-strength tablet containing 160 mg of trimethoprim and 800 mg of sulphamethoxazole) was given three times weekly as primary Pneumocystis carinii (PCP) prophylaxis. Thirty percent (n = 15) of the patients experienced adverse reactions 8-20 (mean 12.7) days after starting with TMP-SMX. At entry, low cysteine and glutathione levels in plasma were found in the HIV-positive patients. Age, sex, CD4+ count, plasma cysteine and glutathione levels were not risk factors for adverse reactions to TMP-SMX. However, concomitant therapy with nucleoside analogues was associated with increased risk for TMP-SMX reactions. Oral NAC 800 mg daily was well tolerated, but replenished neither cysteine nor glutathione levels in plasma. NAC 800 mg/day did not significantly decrease the risk of adverse reactions to TMP-SMX in this study, and could thus not be recommended for this purpose. A prolonged pretreatment period and/or higher dose of NAC may be necessary for clinical effect.

Neutrophil function and glutathione-peroxidase (GSH-px) activity in healthy individuals after treatment with N-acetyl-L-cysteine.

The objective of this study was to evaluate the effect of N-acetyl-L-cysteine (NAC) on neutrophilic functions and as an antioxidant. NAC, 600 mg daily, given orally to healthy individuals for a period of 2 weeks, affected some functions of human neutrophilic granulocytes when tested in vitro. NAC treatment caused a decrease in the production of superoxide anions by stimulated neutrophils and the improvement of their phagocytic capacity although it did not affect their random or chemotactic migration. The level of glutathione peroxidase (GSH-px) in thrombocytes of the NAC-treated individuals was increased in comparison with the activity before treatment. These results suggest that NAC might act as a scavenger of oxygen-derived free radicals released by stimulated neutrophils and thereby protect the tissue against the radical caused injury as well as optimize phagocytosis.

Acyclovir and prednisolone treatment of acute infectious mononucleosis: a multicenter, double-blind, placebo-controlled study.

Ninety-four patients with infectious mononucleosis and symptoms < or = 7 days were randomized to treatment with oral acyclovir (800 mg 5 times/day) and prednisolone (0.7 mg/kg for the first 4 days, which was reduced by 0.1 mg/kg on consecutive days for another 6 days; n = 48), or placebo (n = 46) for 10 days. Oropharyngeal Epstein-Barr virus (EBV) shedding was significantly inhibited during the treatment period (P = .02, Mann-Whitney rank test). No significant effect was observed for duration of general illness, sore throat, weight loss, or absence from school or work. The frequency of latent EBV-infected B lymphocytes in peripheral blood and the HLA-restricted EBV-specific cellular immunity, measured 6 months after onset of disease, was not affected by treatment. Thus, acyclovir combined with prednisolone inhibited oropharyngeal EBV replication without affecting duration of clinical symptoms or development of EBV-specific cellular immunity.

Effect of N-acetylcysteine(NAC) treatment on HIV-1 infection: a double-blind placebo-controlled trial.

OBJECTIVE: In a double-blind placebo-controlled trial, human immunodeficiency virus (HIV)-seropositive patients with a CD4 lymphocyte cell count of more than 200 x 10(6) . l-1 were randomised to receive either 800 mg N-acetylcysteine (NAC) or placebo for 4 months. Before treatment low plasma cysteine levels, high free radical activity in neutrophils in the presence of autologous plasma-measured by the nitroblue tetrazolium (NBT) test- and increased tumor necrosis factor (TNF)-alpha levels were found in the HIV positive patients. RESULTS: After treatment the low plasma cysteine level in the NAC group increased to normal, and the decline of the CD4+ lymphocyte count before the study start, was less steep in the NAC group than in the placebo group after treatment. There was also a reduction in TNF-alpha level. However, NAC had no effect on the radical production by neutrophils, and although it did not increase the CD4+ cell count, it may have decreased the decline in CD4+ cells. CONCLUSION: Further controlled trials with NAC are needed to determine whether it has a beneficial effect in the treatment of asymptomatic HIV-infected individuals.

Oxygen radical release by neutrophils of HIV-infected patients.

Neutrophils from asymptomatic HIV-infected patients have an increased Nitroblue tetrazolium (NBT) reduction, that is an increased production of oxygen radicals. Plasma from these patients can activate normal neutrophils to an increased NBT-reduction and the neutrophil activating factor thus seems to be mainly plasma bound. Further, the patients also have increased levels of plasma malondialdehyde and thus an increased lipid peroxidation. Plasma cysteine levels are low, a sign of increased consumption of antioxidants. Treatment of the asymptomatic HIV-infected patients with N-acetylcysteine corrected the plasma cysteine levels and had some beneficial effects, but did not inhibit the increased radical production by the neutrophils.

Combined treatment of symptomatic human immunodeficiency virus type 1 infection with native interferon-alpha and zidovudine.

Patients with advanced human immunodeficiency virus type 1 (HIV-1) infection who had p24 antigen despite treatment with zidovudine (AZT) for 4-28 months received 3 x 10(6) IU of native interferon-alpha (IFN-alpha) daily for 3 months. Infectious HIV was detected in the plasma of all patients, in most cases at high titers, before IFN-alpha treatment. There was no correlation between HIV titers and p24 antigen levels. Antiviral activity, as measured by significantly decreased levels of infectious virus or p24 antigen, was observed in six of eight completely treated but in only one of nine incompletely treated patients. After termination of IFN-alpha treatment, there was a significant rise of p24 antigen levels. During IFN treatment, absolute CD4 cell counts showed a tendency toward an increased rate of decline. The side effects were unexpectedly severe. Despite its anti-HIV effect in vivo, IFN-alpha in the dosages used does not seem to be a viable additional treatment for severely immunodeficient patients in ongoing AZT therapy.

Epstein-Barr virus-carrying B cells in the blood during acute infectious mononucleosis give rise to lymphoblastoid lines in vitro by release of transforming virus and by proliferation.

In accordance with earlier studies, we detected higher numbers of Epstein-Barr virus (EBV)-carrying lymphocytes (B-EBV) in the blood of acute infectious mononucleosis (IM) patients and higher amounts of transforming EBV particles in the saliva compared to healthy seropositive individuals. B cells grew in cultures seeded with the low and high density IM lymphocytes. The majority of B cells which grew acquired the infection in vitro (2-step outgrowth), because addition of virus neutralizing antibodies considerably reduced the emergence of lymphoblastoid cell lines (LCLs). Only the minority of the explanted B-EBV cells proliferated. The antiviral drug phosphonoformate (PFA) did not influence the frequency of 2-step LCLs in the IM cultures. This may indicate that a large proportion of EBV carrying B cells have already entered the viral productive cycle in vivo and passed the PFA-sensitive stage at the time of explantation. Earlier experiments with blood of healthy seropositive individuals showed an inhibitory effect of PFA on the generation of LCLs. One healthy individual who entered this study as a control, probably had a reactivated EBV infection as judged by the anti-EA activity in his serum and the high level of virus in his saliva. He had antibodies against EBV nuclear antigens (EBNA), and therefore he did not have a primary infection at the time of the test. Judged by the number of wells with B cell growth, the frequency of virus-carrying B cells in his blood was low. It seems that anti-EBV immunity can control the number of infected B cells in the blood, but does not influence the virus load in the epithelial cells.

Influence of human plasma high density lipoproteins from septic patients on different functions of normal human neutrophils.

High Density Lipoproteins (HDL) from three patients with E. coli sepsis contained high, low and no Serum Amyloid Protein (Apo SAA), respectively. Preincubation of neutrophils from healthy persons for half an hour with sepsis HDL as well as normal HDL increased the phagocytosis, the stimulated nitroblue tetrazolium reduction, the chemotaxis and the random migrations of these cells. However, for all these functions, lower values were obtained after incubation with sepsis HDL containing high amounts of apo SAA than with normal HDL. A qualitative change of HDL might thus in part be responsible for the decreased function of neutrophils noted during the acute phase of bacterial infections.

Plasma clearance of intravenous fat emulsion in acute bacterial infections.

A variety of abnormalities in lipid metabolism during infection have been reported and it has been questioned whether intravenous fat emulsion should be given in septic states. This study was designed to investigate the removal of a fat emulsion from plasma during septicaemia. An intravenous fat tolerance test (0.1 g of fat in the form of Intralipid 10% per kg b.w.) was performed in 28 adult patients who had been admitted for suspected septicaemia. All patients were febrile (mean body temperature 38.9 +/- 0.1 degrees C SEM), the majority had leucocytosis (mean value 10.5 +/- 1.0 10 (9)1 ) and all were haemodynamically stable. On the basis of bacterial cultures taken from various locations, the patients were classified as septic (n = 12; gram-negative sepsis, n = 8; gram-positive sepsis, n = 4), infected but without septicaemia (n = 9) or febrile of unknown cause (all cultures negative, n = 7). Plasma fractional removal rates of intravenous fat were similar in the different groups. The mean plasma clearance rate of intravenous fat was 6.6 +/- 0.5 (% minute), which does not differ from that reported for healthy controls. All patients with proven septicaemia and 5 non-septic patients were studied two weeks later when their infection had been treated and they were off antibiotic medication. The plasma clearance rate of i.v. fat was significantly lower in the recovery phase than during the acute febrile state (5.9 +/- 0.5 vs 4.2 +/- 0.4 %/min, n = 17; p < 0.05). It is concluded that clearance of intravenous fat emulsions from plasma is unimpaired in haemodynamically stable patients with septicaemia or other acute infections.

Increased production of malondialdehyde in patients with HIV infection.

The mean plasma content of malondialdehyde (MDA) in 30 patients in different stages of HIV infection was found to be about 30% higher than that in controls. The phenomenon was not correlated to the degree of immunodeficiency and was noted early in the course of the disease. This indicates a higher degree of basal lipid peroxidation, which might contribute to the tissue damage seen in these patients. A new reverse phase liquid chromatography method was used for quantitative measurements of MDA in plasma after reaction of this compound with thiobarbituric acid.

Acyclovir treatment in infectious mononucleosis: a clinical and virological study.

Fifty-six patients with a clinical and laboratory diagnosis of infectious mononucleosis who had not been ill for more than seven days, were randomised for peroral treatment with acyclovir (800 mg five times daily) or placebo for seven days in a double blind trial. Clinical, virological and immunological parameters were monitored in each patient for six months. During treatment, shedding of Epstein-Barr virus' as assessed in 36 patients, was significantly reduced (p less than 0.001). However, virus production in the oropharynx returned to pre-treatment levels one week after the cessation of therapy. Virus was detected in 35 patients at enrollment and in 28 of 36 patients at the six-month control. No effect on the clinical course of the disease was noticed. The virus-specific antibody response was also unaffected. A significant reduction in spontaneous outgrowth of in vivo Epstein-Barr virus-infected B-lymphocytes was found at 180 days after treatment in four acyclovir-treated patients compared to six controls (p less than 0.001). In another three patients with over-whelming clinical symptoms causing airway obstruction and/or disseminated intravascular coagulopathy, treatment with intravenous acyclovir (10 mg/kg three times daily) was combined with prednisolone (0.7 mg/kg daily) for ten days. Virus shedding ceased transiently during treatment, but returned to initial levels within one week. A dramatic clinical effect on the pharyngeal oedema and general health of the two patients with airway obstruction was noticed, but was much less evident in a patient with intravascular coagulopathy.