Acceptability to patients of a honey dressing for non-healing venous leg ulcers.

OBJECTIVES: This four-centre feasibility study was undertaken to determine whether Medihoney, a proprietary blend of honeys, is an acceptable treatment for patients with leg ulcers in terms of pain relief, odour control and overall patient satisfaction. METHOD: Forty patients whose leg ulcers had not responded to 12 weeks of compression therapy were recruited. Medihoney dressings were applied on their ulcers for the 12-week study period. All other aspects of their care, including the use of compression bandaging, remained unchanged. Their leg ulcers were assessed every two weeks. This included the use of a patient questionnaire. RESULTS: Overall, ulcer pain and size decreased significantly and odorous wounds were deodorised promptly. This had a positive impact on patient satisfaction with the Medihoney treatment. CONCLUSION: The results support the previously reported positive effects of honey and revealed a high patient acceptance for this treatment. Following these results, comparative clinical trials, which should also consider pain, are now recommended.

PCR-based quantification of Pneumocystis carinii in in vitro systems.

In many laboratories, PCR has become a routine method for the sensitive diagnosis of Pneumocystis carinii in patient samples. In contrast, quantification of fungal numbers in in vitro setups still largely relies on more conventional procedures such as histological stainings. These are time consuming and their applications are limited when dealing with small fungal numbers contaminated with tissue and cellular debris. This study presents a sensitive and rapid method for P. carinii quantification based on PCR analysis that can be easily integrated into standard detection procedures without requiring any major additional steps. P. carinii-specific PCR performed with total DNA extracted from both standard samples with known fungal numbers and experimental samples was quantified relative to PCR products of a standard concentration from a control plasmid added prior to DNA extraction. This measure controlled for variations in DNA extraction and PCR efficiency among the samples to be compared. The correlation between analyzed P. carinii-specific DNA and the actual fungal numbers employed was highly significant.

The hepatitis B virus X protein transactivates viral core gene expression in vivo.

The function of the X protein in the life cycle of mammalian hepadnaviruses is unclear. Based on tissue culture experiments it has been suggested that this protein represents a transcriptional transactivator which might be essential for the expression of the viral core gene. Here we have examined whether the activity of the human hepatitis B virus (HBV) core gene in vivo depends on X coexpression. To this end we compared core gene expression between four lineages of transgenic mice carrying the HBV core gene in cis arrangement with the X gene (cex lineage) and six lineages containing a modified construct in which the start codon of the X gene had been deleted (ce lineage). Whereas all cex lineages consistently exhibited a high-level hepatic core gene expression, the liver-specific core gene expression pattern of the ce lineages was heterogenous with four lineages virtually not expressing the core gene. This defect was due to a strongly reduced transcription since no core mRNA could be detected by Northern blotting. To test whether core gene expression could be restored by providing an intact X gene in trans, we crossbred mice of two lines which expressed no core mRNA or core protein with transgenic mice expressing the X-gene product under the transcriptional regulation of the liver-specific major-urinary-protein promoter/enhancer (MUP-X mice). The introduction of the MUP-X transgene induced core mRNA expression and core protein biosynthesis in the livers of the double-transgenic mice. This demonstrates that the X-gene product has the capacity to transactivate HBV core gene expression in vivo.

Effect on parasite eradication of Pneumocystis carinii-specific antibodies produced in the presence or absence of CD4(+) alphabeta T lymphocytes.

The contribution of specific antibodies (Ab) to successful clearance of Pneumocystis carinii from host pulmonary tissues has received increasing attention. Sera collected from diseased recombinase-activating gene (RAG)-1(-/-), TCRbetaxdelta(-/-), TCRbeta(-/-) and Abeta(-/-) mutants as well as from aerogenic parasite-exposed (aero) and intranasally (i. n.) infected C57BL/6 mice were transferred to RAG-1(-/-) mutants inoculated with freshly isolated parasites. All sera, except for RAG-1(-/-) serum, contained P. carinii-specific Ab of varying isotype concentrations. Four weeks after serum treatment pulmonary parasite numbers were reduced slightly by Abeta(-/-) and C57BL/6-aero sera, and markedly by TCRbeta(-/-) and C57BL/6-i.n. sera. Our data reveal: (1) T cells are essential, and CD4(+) T cells are important for formation of protective Ab; (2) at least in the absence of alpha beta T cells, gamma delta T cells provide help for protective Ab. In vitro treatment of bronchoalveolar lavage cells with the different sera largely led to comparable results. Opsonizing Ab impeding parasite attachment to host cells, as well as Ab possibly neutralizing parasite-secreted products were implicated. Furthermore, serum components other than Abappear to participate in resistance to fungal manifestation.

Pneumocystis carinii pneumonia in mutant mice deficient in both TCRalphabeta and TCRgammadelta cells: cytokine and antibody responses.

Resistance to Pneumocystis carinii is achieved through cell-mediated and humoral immunity, but the interplay between these two systems in the immunocompetent host is not fully understood. TCRbetaxdelta-/- double-mutant mice deficient of all T cell populations naturally acquired P. carinii pneumonia with lethal consequences. Moribund mutants displayed numbers of pulmonary pathogens comparable to RAG-1-/- mice lacking all functional T and B lymphocytes. Pulmonary lavage cells of diseased TCRbetaxdelta-/- mutants secreted proinflammatory cytokines tumor necrosis factor-alpha, interleukin (IL)-12, and interferon-gamma, but not IL-4, -5, or -10. Serum immunoglobulin levels of both healthy and diseased mice were significantly reduced compared with immunocompetent animals. Secreted antibodies were mainly IgM, which also bound P. carinii. Mutants completely lacked IgG1, emphasizing strict T cell dependence of immunoglobulin switching to this isotype. Other IgG subclasses were strongly reduced and did not bind P. carinii. These results suggest that T cells are crucial for generation of antibodies against P. carinii relevant to resistance.

The hepatitis B virus e antigen cannot pass the murine placenta efficiently and does not induce CTL immune tolerance in H-2b mice in utero.

The function of the secretory core gene product (HBeAg) of the human hepatitis B virus is unclear. It has been discussed that this protein may be passed from the mother to the fetus, where it might induce immunologic tolerance. Here we have examined this possibility with transgenic mice expressing high levels of HBeAg. Analysis of serum samples obtained from nontransgenic fetuses which developed in HBeAg-positive mothers showed no evidence that the HBeAg can pass the placenta. Moreover, direct examination of the HBeAg- and HBcAg-specific cytotoxic T-cell immune response of H-2b mice which developed in either transgenic or nontransgenic mothers revealed no indication that mice which could have been exposed to the HBeAg in utero become tolerant to HBV core gene products. From these data we conclude that the placenta represents an efficient barrier for HBeAg transfer and that the HBeAg does not tolerize cytotoxic T cells, at least in mice of the H-2b haplotype.

Pneumocystis carinii and the immune response in disease.

Pneumocystis carinii causes serious disease in immunocompromised patients, notably in AIDS patients. Our understanding of the biology of this fungus is advancing but still incomplete. Protective immunity against P. carinii is mediated by CD4+ T cells in the immunocompetent host, but precise resistance mechanisms remain to be determined.

Activated pulmonary macrophages are insufficient for resistance against Pneumocystis carinii.

CD4+ T cells are pivotal for elimination of Pneumocystis carinii from infected lungs, and alveolar macrophages are considered the main effector cells clearing the infected host of P. carinii organisms. To investigate this issue, several mutant mouse strains were used in a previously established experimental setup which facilitates natural acquisition of disease through inhalation of airborne fungal organisms. Mutant mice deficient in major histocompatibility complex class II molecules (A beta(-/-)), T-cell receptor alphabeta cells (TCR beta(-/-)), or all mature T and B lymphocytes (RAG-1(-/-)) were naturally susceptible to P. carinii, whereas mouse mutants lacking the gamma interferon (IFN-gamma) receptor (IFN-gamma-R(-/-)) or tumor necrosis factor alpha (TNF-alpha) type I receptor (p55) (TNF-alpha-RI(-/-)) resisted disease acquisition. Analysis of pulmonary cytokine patterns and free radical expression revealed the presence of superoxide, nitric oxide, and interleukin-1 (IL-1) mRNA and elevated levels of IFN-gamma, TNF-alpha, and IL-12 in diseased TCR beta(-/-) and RAG-1(-/-) mice. Pulmonary macrophages of all diseased mouse mutants expressed scavenger and mannose receptors. Morbid A beta(-/-) mutants displayed significant NO levels and IL-1 mRNA only, whereas heterozygous controls did not exhibit any signs of disease. Interestingly, neither IFN-gamma nor TNF-alpha appeared to be essential for resisting natural infection with P. carinii, nor were these cytokines sufficient for mediating resistance during established disease in the absence of CD4+ T lymphocytes. Taken together, the results indicated that an activated phagocyte system, as evidenced by cytokine and NO secretion, in diseased mutants was apparently operative but did not suffice for parasite clearance in the absence of CD4+ TCR alphabeta cells. Therefore, additional pathways, possibly involving interactions of inflammatory cytokines with CD4+ T lymphocytes, must contribute to successful resistance against P. carinii.

Naturally acquired Pneumocystis carinii pneumonia in gene disruption mutant mice: roles of distinct T-cell populations in infection.

When kept under strict specific-pathogen-free conditions, H-21-Abeta (Abeta(-/-),T-cell receptor beta (TCRbeta(-/-)), and recombinase-activating gene 1 (RAG-1(-/-) gene disruption mutant mice, deficient in conventional CD4+ T cells, TCRalphabeta cells, and all peripheral T and B lymphocytes, respectively, consistently developed lethal Pneumocystis carinii pneumonia through natural infection. The most severe symptoms appeared in RAG-1(-/-) mutants. In contrast, TCRdelta(-/-) and beta2-microglobulin(-/-)(beta2m-/-) mutants, deficient in TCRgammadelta cells and conventional CD8alphabeta+ TCRalphabeta cells, respectively, were fully resistant to infection. Our data indicate not only the insufficiency but also the dispensability of CD8 alphabeta+TCRalphabeta cells and of TCRgammadelta lymphocytes in resistance to P. carinii infection. Under disease conditions, large numbers of unusual single-positive CD4+ and CD8alphabeta+ as well as double-negative TCRgammadelta subpopulations of cells accumulated in lungs of TCRbeta(-/-) mutants. This accumulation was consistently accompanied by a drastic increase in the pulmonary B-cell population. In contrast, CD8alphabeta+ TCR alpha beta cells, but no B cells, appeared in lungs of parasitized Abeta (-/-) mutants. Since lung damage and parasite numbers were less prominent in morbid TCRbeta(-/-) and Abeta(-/-) mutants than in diseased RAG-1(-/-) mice, the remaining lymphocytes accumulating in lungs of the former two mutants seem to perform residual resistance functions.

T- and B-lymphocyte-independent formation of alveolar macrophage-derived multinucleated giant cells in murine Pneumocystis carinii pneumonia.

Multinucleated giant cells developed in Pneumocystis carinii-diseased gene disruption mutant mice deficient in major histocompatibility complex class II molecules, T-cell receptor alpha beta cells, or all mature T and B lymphocytes. These findings demonstrate lymphocyte-independent fusion of alveolar macrophages under morbid conditions. Pulmonary parasite burden seems to be a decisive factor in multinucleation.

Nitric oxide production and mycobacterial growth inhibition by murine alveolar macrophages: the sequence of rIFN-gamma stimulation and Mycobacterium bovis BCG infection determines macrophage activation.

Prestimulation with recombinant-interferon-gamma (rIFN-gamma) followed by Mycobacterium bovis BCG infection induced high nitric oxide production and potent mycobacterial growth inhibition in murine alveolar macrophages. Reversal of the sequence of treatments caused opposite effects, suggesting that a sequential 2-step process comprising first rIFN-gamma stimulation and second mycobacterial infection is operative in the activation of alveolar macrophages.

Above- and below-ground environmental influences on leaf conductance ofCeanothus thyrsiflorus growing in a chaparral environment: drought response and the role of abscisic acid.

Small shrubs ofCeanothus thyrsiflorus were grown in 19-1 pots irrigated under natural conditions in a chaparral region of Southern California and then subjected to soil drying. Characteristics of leaf gas exchange, leaf water potential, and concentrations of the stress hormone abscisic acid in the xylem sap, ABAxyl, were determined at various stages of drought. Diurnal changes in conductance were strongly correlated with leaf net photosynthesis rate, which provides an effective, integrative predictor of above-ground climate effects on conductance. In drought conditions, ABAxyl concentration increased. Increases in the concentration range of 50-500 nmol/l appeared to induce stomatal closure, restricting water loss and carbon dioxide uptake. When the momentary water potential is related to ABAxyl, ABA appeared to increase significantly only after a threshold of approximately -1.5 MPa was exceeded. At less negative water potentials, large variation in ABAxyl in the 50-1000 nmol/l range occurred for all water-potential values, because ABAxyl remains relatively constant over diurnal courses as water potentials decrease and then recover. When the water potential became more negative than -1.5 MPa, ABAxyl concentrations occurred between approximately 500 and 10 000 nmol/l and even greater in isolated cases. An approximately linear relationship is recognizable between ABAxyl and momentary water potential in this range because in plants under drought conditions, ABAxyl increases during the course of the day as water potential decreases. Increases in ABAxyl in the high concentration range were associated with relatively minor additional restrictions in gas exchange, but they might contribute to improved water use efficiency and explain diurnal changes in the potential for stomatal opening that have been observed in Mediterranean sclerophyllous species. When we examined long-term seasonal change in the response of irrigated plants, changes in average daily temperature greater than 10°C occurred (also associated with shifts in relative humidity and radiation input), which apparently led to small changes in predawn water potential in the -0.1 to -0.7 MPa range. Increases in ABAxyl occurred that were in turn negatively correlated with daily maximum leaf conductance. Thus, chaparral shrubs under non-drought conditions seem to sense even small changes in environmental conditions, in our opinion most probably due to initial drying of the uppermost soil and synthesis of ABA in the shallow roots. The results support the hypothesis that information of photosynthesis rate and predawn water potential may be used as primary variables to predict canopy conductance of Mediterranean sclerophyll shrub vegetation.