In vitro prevention of vaccinia and herpesvirus infection spread in explanted human corneas by N-chlorotaurine.

OBJECTIVE: Weak oxidants produced by activated human leukocytes are proven antimicrobial substances. We tested whether N-chlorotaurine (NCT, taurine chloramine), the chlorinated metabolite of the amino acid taurine, in addition to direct virucidal effects on viral suspensions, has the capability to prevent cell-to-cell spread of viruses in human corneal epithelium. METHODS: Human corneal grafts were infected in vitro with poxvirus (vaccinia virus, VV) and herpesvirus. Different NCT dilutions were added to prevent viral spread within the corneal epithelium as detected by immune-staining and microscopy of cytopathic effects. Additionally, virus release was measured by cell culture. RESULTS: Addition of NCT significantly reduced the number of VV-infected epithelial cells at concentrations as low as 0.01% in culture medium, which was far beyond cytotoxic concentrations in long-term cultures. The release of virus by the infected corneal grafts was reduced by 2-3 log(10 )as well. As expected, herpesvirus infection was also positively affected. CONCLUSION: Smallpox has been known as a major cause of blindness in historical outbreaks. NCT could therefore provide an additional supportive means for treating orthopoxvirus-associated keratitis. Additionally, biocompatible local antiseptics like NCT could also serve as an experimental treatment in other keratitis of suspected viral origin.

Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.

BACKGROUND: The detection of a broad range of bacteria by PCR is applied for the screening of blood and blood products with special attention to platelet concentrates. For practical use it is desirable that detection systems include Gram-positive, Gram-negative and non-Gram-stainable bacteria. It is quite challenging to achieve high sensitivity along with a clear negative control with PCR reagents, because especially Taq polymerase is contaminated with traces of bacterial DNA. METHODS: Bacterial DNA decontamination of Taq polymerase was attempted by two different methods using the restriction enzyme Sau 3A1 and microfiltration. Additionally a commercially available Taq polymerase depleted of bacterial DNA was included. A published real-time PCR specific for Gram-negative bacteria was adapted for Gram-positive bacteria, including certain Staphylococcus species and Mycobacteria, and was used to charge the three Taq polymer-ases depleted of bacterial DNA contamination RESULTS: Despite published reports about successful DNA decontamination, all three approaches performed poorly in experiments done in this study. Sensitivity ranged at approximately 50-100 colony forming units (CFU) per PCR reaction for Escherichia coli and Staphylococcus epidermidis, corresponding to 1,250-2,500 CFU/ml sample material. Conclusion: It seems unsatisfying to accept detection limits that high for diagnostic bacterial PCR even if highly multiplexed. Reliable methods for DNA decontamination of Taq polymerase are needed and would present one important step towards bacterial DNA detection with high sensitivity.

Involvement of protein kinase C in phagocytosis of human retinal pigment epithelial cells and induction of matrix metalloproteinase secretion.

Protein kinase C (PKC) is involved in cell activation. We investigated PKC-mediated pathways and secretion of matrix metalloproteinases (MMPs) in phagocytosis by human retinal pigment epithelial cells (RPE). We used time-resolved fluorometry for europium-labeled microsphere uptake and gel zymography to assay the influence of PKC modulators. PKC inhibitors blocked phagocytosis by RPE. ARPE-19, a human RPE-cell line, showed reduced secretion of MMP-2, although MMP-9 secretion by PKC activation was conserved in both cell types, namely in the primary RPEs and in the RPE-cell line. Particle uptake by RPE cells requires activation of PKC; the use of PKC inhibitors as new anticancer drugs may possibly cause ocular side-effects.

Characterization of antigen-presenting cells in fresh and cultured human corneas using novel dendritic cell markers.

PURPOSE: Adult healthy human corneas bear a distinctive number of antigen-presenting cells (APCs) important for the fate of a graft. The purpose of this study was to differentiate between Langerhans cells (LCs) and other dendritic cells (DCs) and between mature and immature APCs in fresh and cultured human corneas using specific markers. METHODS: Immunofluorescence double staining was performed for Langerin/CD207, CD1a, DC-SIGN/CD209, DC-LAMP/CD208, CD45, CD11c, CD11b and HLA-DR. RESULTS: Langerin(+)/CD1a(+)/HLA-DR(+) LCs (approximately 100 cells/mm(2) in fresh corneas) were found in the limbal and peripheral regions of corneal epithelium and the anterior stroma up to 83 days of culture. All these cells coexpressed CD45 and CD11c. DC-SIGN(+)/CD45(+) DCs (approximately 150 cells/mm(2) in fresh corneas) were detected mainly peripherally and in the anterior stroma, even in long-term cultured corneas. Most of these cells were HLA-DR(-). Few mature DCs (DC-LAMP(+)/HLA-DR(+)) were found in fresh and cultured corneas. Macrophages (CD11c(-)/CD11b(+)) were seen in the peripheral, paracentral, and even central regions of the posterior stroma. CONCLUSIONS: This is the first demonstration that human corneas harbor populations of Langerin(+)/CD1a(+)/HLA-DR(+) LCs and DC-SIGN(+) DCs in a distribution pattern similar to that in the skin. Few APCs are in a mature state (DC-LAMP(+)). Given the reduced but not complete depletion of APCs during organ culture, these grafts still bear a potential risk for rejection.

N-chlorotaurine is an effective antiviral agent against adenovirus in vitro and in the Ad5/NZW rabbit ocular model.

PURPOSE: To determine whether N-chlorotaurine (NCT) demonstrates antiviral activity against adenovirus (Ad) in vitro and in the Ad5/NZW rabbit ocular model. METHODS: The in vitro activity of NCT was evaluated by incubating different Ad serotypes with several concentrations of NCT for 1 hour and determining the reduction in Ad titers. In rabbit study 1, Ad5-infected eyes were treated with 2.5%, 2.0%, and 1.0% NCT; 0.5% cidofovir; or saline. NCT and saline groups were treated 10 times for 1 day and then 5 times daily for 6 days. In rabbit study 2, Ad5-infected eyes were treated with 1.0% NCT/0.1% ammonium chloride (NH4Cl), 0.1% NCT/1.0% NH4Cl, 0.1% NCT/0.1% NH4Cl, and 0.5% cidofovir or saline. The NCT and saline groups were treated five times daily for 10 days. Cidofovir-treated eyes received the authors' standard cidofovir dose regimen: twice daily for 7 days. RESULTS: In vitro, NCT demonstrated concentration-dependent direct inactivation of all ocular Ad serotypes tested. Rabbit study 1: 2.5%, 2.0%, 1.0% NCT, and cidofovir demonstrated significantly fewer positive cultures per total cultures during days 1 to 14, compared with saline. Rabbit study 2: 1.0% NCT/0.1% NH4Cl, 0.1% NCT/1.0% NH4Cl, 0.1% NCT/0.1% NH4Cl, and cidofovir demonstrated significantly fewer positive cultures per total cultures, during days 1 to 14; shorter durations of shedding; and lower mean combined titers, during days 7 to 14, compared with saline. Cidofovir was significantly more effective than NCT in several outcome measures in both rabbit studies. CONCLUSIONS: NCT demonstrated antiviral activity against adenovirus in vitro and in vivo. Further development of NCT as a topical antimicrobial is indicated.

Functional T-cell anergy in a case of persistent polyclonal B-cell lymphocytosis.

The T-cell population of a patient with persistent polyclonal B-cell lymphocytosis (PPBL) presenting with an intermittent Epstein-Barr virus (EBV)-associated disease was studied. Unstimulated T-cells did not express CD40 ligand (CD40L), whereas activation with IL-2 led to expression of this costimulatory molecule. CD40L expression was inhibited upon incubation with the supernatant of an EBV-positive B-cell line (SM) which had been grown spontaneously from the patient's peripheral blood cells. The supernatant of SM cells effectively inhibited cytotoxic T-cells. Elevated levels of IL-10, TNF-alpha and soluble CD40 were found in the supernatant of SM cells. Additionally, enhanced levels of LMP-1 protein were detected.

Investigation of bacterial and viral agents and immune status in Behcet's disease patients from Iran.

AIM: Behçet's disease (BD) is an autoimmune disorder associated with HLA-B51 positivity. Serologic/genomic findings have suggested microbes as possible causative agents and the geographical distribution suggests environmental influences. METHODS: We performed comparative analyses of 40 patients with BD or related symptoms not fulfilling BD criteria. Patients originating from different regions of Iran were tested by molecular/serological methods for human herpes viruses and parvovirus B19, two Chlamydiae species, as well as Coxiella, Listeria, Yersinia, Leptospira and Mycobacterium paratuberculosis. Human leukocyte antigen-typing was performed: testing of cytokine profiles and immune mediators representative for the cellular immune system, including neopterin/kynurenine production. RESULTS: No apparent differences in interleukin (IL)-4, 6, 8 and 10 were observed, whereas production of soluble IL-2-receptor and tumor necrosis factor (TNF)-alpha were more pronounced in the BD group. Neopterin/kynurenine production was comparable, although both groups showed twice the levels of healthy people. No significant differences of herpes simplex virus (HSV) antibody titres were observed but higher titres against Chlamydophila pneumoniae were found in the controls. In 20 BD patients and controls neither parvovirus B19 DNA was detected nor bacterial DNA. Viral DNA of Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpes virus (HHV)8 was detected more frequently in the BD group, whereas HSV DNA was only found in the controls, indicating that stomatitis might be caused by HSV. CONCLUSION: Although no significant association of BD was detected with a single pathogen, our findings suggest that detection of HSV DNA or Chlamydiae would rather argue against classic BD. Whether there is a discriminative potential of the tested immune mediators/receptors has to be elucidated in further studies.

Diazepam leads to enhanced severity of orthopoxvirus infection and immune suppression.

Benzodiazepines are drugs widely used as tranquilizers and in various other indications. We treated Balb/c mice with diazepam and infected them with cowpox (CPXV) and vaccinia virus (VACV). Disease index, weight loss and the antibody response were determined. Additionally the influence of different benzodiazepines on the mitogen response of human peripheral blood lymphocytes and spleen cells was tested. Diazepam led to earlier disease onset, prolonged duration of symptoms, higher weight loss and overall disease index in VACV infected mice. CPXV infected mice developed poxviral skin lesions only after drug administration and a significant decrease in the specific antibody response was also observed. Diazepam and alprazolam also inhibited the proliferative response of human lymphocytes/spleen cells in vitro but did not show noteworthy apoptotic effects. It is surprising that even a single dose of diazepam has a profound influence on the immune system, sufficient to facilitate symptomatic infectious disease. These data provide first evidence that commonly used drugs like Valium may augment severity of rare poxvirus infections such as CPXV or monkeypox. As VACV is still used as life vaccine against smallpox there is also a risk of enhanced side effects or possible interference with the success of vaccination.

Active in vitro reduction of antigen presenting cells in human corneal grafts using different chemokines.

BACKGROUND: For the fate of a graft the antigen presenting cells play an important role. Chemokines can lead to enhanced migration of these cells. We therefore investigated if fresh human corneas bear the chemokine receptor 7 (CCR7) and if its ligands can force the emigration of dendritic cells in an in vitro model. METHODS: We used human corneas excluded for transplantation and performed migration tests using chemokine ligands 19 (CCL19) and 21 (CCL21) or the complement factor 5a (C5a). Emigrated cells were collected up to 35 days and stained by immunofluorescent double labeling in triple layer technique with Langerin/CD207, DC-SIGN/CD209, CD14, and HLA-DR. In parallel, fresh and cultured human corneas were stained for CCR7. RESULTS: We found in fresh human corneas, as well as in long-term cultured ones, a low CCR7 expression that nearly diminished after 28 days. In vitro Langerhans cell emigration could be enhanced only by CCL19, whereas dendritic cells were strongly influenced by CCL19, CCL21, and C5a. HLA-DR(+) cells showed numerically the highest in vitro emigration rate. Macrophages/monocytes were not influenced by the used chemokines. CONCLUSIONS: Although human corneas reduce their antigen presenting cells numbers during long-term culture, this effect could be significantly enhanced by using chemokines.

Phagocytosis of human retinal pigment epithelial cells: evidence of a diurnal rhythm, involvement of the cytoskeleton and interference of antiviral drugs.

Retinal pigment epithelial (RPE) cells provide crucial functions for the maintenance of the retinal environment. We investigated the phagocytotic mechanisms of RPE cells evaluating the question whether particle uptake underlies a diurnal rhythm. Additionally, a possible connection of volume regulation and the phagocytotic function of RPE cells was studied. As antiviral nucleoside analogues influence cell-volume-regulating mechanisms, we tested several antiviral drugs. Cultured primary RPE cells and a permanent cell line (ARPE-19) were tested for uptake of europium-labeled microspheres quantified by time-resolved fluorometry. Cells were also exposed to cyclic illumination or continuous light and dark culture conditions. Inhibitors of cytoskeleton (microtubuli, actin) and osmotic swelling were also tested. Ingested FITC-labeled microparticles were found in phagosomes strongly associated which the cytoskeleton as they could not be easily moved by laser tweezer microscopy. Phagocytosis was observed predominately during dark intervals and was reduced by continuous light exposure. The diurnal rhythm of unsynchronized RPE cultures was abolished by microtubule inhibitors although no inhibition of overall particle uptake by cytoskeletal blockers was observed. Hypoosmotic swelling of RPE also decreased phagocytosis. Acyclovir was found inhibitory in ARPE-19 cells, whereas azidothymidine showed a protracted inhibiting activity on primary RPE cells and ganciclovir was inactive in both cell types. The presence of a diurnal rhythm also in culture indicates genetic determination of light-regulated particle uptake. This mechanism appears to be influenced by the regulation of cell volume and microtubule function. Inhibition of RPE function by antiviral drugs is a novel finding and in accordance with interferences of the tested drugs with cellular chloride channels described earlier. It may give a hint towards possible ocular side effects in the long-term use of nucleoside-analogous substances.

Retinal pigment epithelial phagocytosis and metabolism differ from those of macrophages.

The purpose of this study is to compare primary human retinal pigment epithelium (RPE) cells with respect to particle uptake and further processing steps with immunological phagocytes for a better understanding of the possible role of RPE cells in triggering autoimmune diseases in the eye. We investigated the similarities of human RPE and monocytes/macrophages studying the uptake of fluorescein- and europium-labeled synthetic microparticles and microbial pathogens by human and bovine RPE cultures and a permanent RPE cell line (CRL). The uptake was monitored by laser scanning microscopy, flow cytometry and time-resolved fluorescence analysis; for comparison, macrophages and a macrophage-like cell line (MonoMac6) were used. A size-dependent uptake was seen in primary RPE cultures as well as in CRL, showing a preferential uptake of smaller beads followed by Staphylococcus aureus and Escherichia coli. Opsonization with serum caused a modest increase in bacteria uptake, but in contrast to macrophages, the classical complement receptors were not found on RPE cells. Living bacteria were also ingested in a time-dependent manner, but, as no intracellular overgrowth was observed, we further investigated the oxidative ability of RPE as a possible mechanism for microbial suppression. Unlike macrophages/granulocytes, no respiratory burst was detected in RPE cells, but, comparable to MonoMac6, IFN-gamma induced neopterin in the human RPE. Interestingly a diurnal rhythm of phagocytosis was observed which was influenced by light exposure suggesting that RPE cells maintain their circadian rhythm also in cell culture to a certain extent. This study further demonstrates that in addition to similar phagocytic properties the RPE still shows substantial metabolic differences in comparison to blood-derived phagocytes.