Renal allograft recipients fail to increase interferon-γ during invasive fungal diseases.

Invasive fungal diseases are a major cause of death in renal allograft recipients. We previously reported that adjunctive recombinant human interferon-γ therapy has clinical utility for invasive fungal diseases after renal transplantation. We have now developed a rapid peripheral blood-based quantitative real-time PCR assay that enables accurate profiling of cytokine imbalances. Our preliminary studies in renal transplant patients with invasive fungal diseases suggest that they fail to mount an adequate interferon-γ response to the fungal infection. In addition, they have reduced IL-10 and increased TNF-α when compared to stable renal transplant patients. These preliminary cytokine profiling-based observations provide a possible explanation for the therapeutic benefit of adjunctive human interferon-γ therapy in renal allograft recipients with invasive fungal diseases.

Advanced Cancer Patients' Prognostic Awareness and Its Association With Anxiety, Depression and Spiritual Well-Being: A Multi-Country Study in Asia.

AIMS: The associations between prognostic awareness, acceptance of illness and psychological outcomes (anxiety, depression and spiritual well-being) remain unclear. This study examined the associations between prognostic awareness and various psychological outcomes and how they can be moderated by patient acceptance of illness (cancer). MATERIALS AND METHODS: In total, 1184 patients with stage IV solid cancer were recruited at major public hospitals across four Asian countries (China, India, Sri Lanka, Vietnam). Prognostic awareness and acceptance of illness were assessed through self-reported understanding of treatment intent and acceptance of illness, respectively. Anxiety and depression were assessed using the Hospital Anxiety and Depression Scale, whereas spiritual well-being was measured using the Functional Assessment of Chronic Illness Therapy - Spiritual Well-Being Scale. Multivariate regressions were used to estimate the associations while controlling for patient characteristics. RESULTS: Compared with being unaware of their prognosis (i.e. believing that their cancer is curable), being aware or unsure of their prognosis was associated with higher anxiety and depressive symptoms, and lower spiritual well-being scores. Acceptance of illness moderated these relationships and improved the psychological outcomes. CONCLUSIONS: The results suggest that disclosure of prognostic information should be provided in conjunction with psychological interventions that focus on acceptance of illness.

Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy.

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Exogenous interferon-gamma immunotherapy for invasive fungal infections in kidney transplant patients.

The incidence of invasive fungal infections (IFIs) in nonneutropenic solid organ transplant patients is increasing. We report our clinical experience with the use of interferon-gamma (IFN-gamma) immunotherapy in seven renal transplant patients who developed life threatening, disseminated IFIs refractory to conventional antifungal drug therapy. The infections were all microbiologically and histologically proven. The rapid cure of these disseminated infections with exogenous IFN-gamma injections was not associated with impaired kidney allograft function despite the use of liposomal amphotericin B in all cases. No clinical toxicity from the IFN-gamma immunotherapy was seen and no IFI relapsed during long-term follow-up. Our experience is both uncontrolled and in patients with unpredictable fungal infection-related outcomes. However, compared to standard approaches, the accelerated cure of life threatening, disseminated IFIs with 6 weeks of combination antifungal drug therapy and IFN-gamma immunotherapy saved lives, retained allograft function and led to substantial cost savings in this small patient group.

Progesterone receptor reactivity in renal oncocytoma and chromophobe renal cell carcinoma.

AIMS: To investigate the reactivity for oestrogen and progesterone receptors (ER and PR) in renal oncocytoma (RO) and chromophobe renal cell carcinoma (CHRCC). MATERIALS AND METHODS: Thirty-eight RO, 25 CHRCC, 20 papillary RCC with oncocytic cytoplasm and 10 clear cell RCC with dominant eosinophilic cytoplasm were submitted for immunohistochemistry for ER, PR, CD117 and RCC. RESULTS: All cases of RO and CHRCC displayed moderately positive reactivity for PR. The nuclear reactivity ranged from 60% to 90% in RO and from occasional cells to 70% in CHRCC. In CHRCC, reactivity tended to be more prevalent in areas of tumour cells with eosinophilic cytoplasm. Progesterone reactivity was focal in areas. All RO and most CHRCC were reactive for CD117 and neither RO nor CHRCC was reactive for RCC. CD117 reactivity tended to be more intense in CHRCC than in RO. Negative reactivity for CD117 and positive reactivity for RCC were observed in almost all RCC, as reported in the literature. CONCLUSIONS: PR can be used in combination with CD117 and RCC in the differential diagnosis of RO and eosinophilic variant of CHRCC with other RCC with oncocytic or eosinophilic cytoplasm.

Lower limb dominance and volume in healthy individuals.

Upper limb dominance is associated with increased limb volume, however there is a paucity of evidence if this is true for the lower limbs. This study investigated if there is a normative volume difference between the dominant and nondominant leg. Healthy volunteers between the ages of 18-40 years were recruited. Exclusion criteria included previous lower limb surgery, BMI >30, or pregnancy. An experienced lymphedema nurse specialist measured the circumference of each limb at 4 cm intervals from the malleolus to the groin. Measurements were used to calculate volume of each limb in milliliters. 100 (52 male, 48 female) participants met our inclusion criteria. 86% were right leg dominant and 14% left leg dominant. 93% demonstrated an average increased volume of 349 ml (4.5%) in the dominant leg which is statistically significant (p<0.001). Age, sports, and gender did not affect lower limb volumes. This is the first study to show a normative variance in leg volume in healthy individuals, with a greater volume in the dominant leg. This should be taken into consideration when managing and measuring outcomes for patients with conditions resulting in enlarged lower limbs.

Reduction of the viral load of HIV-1 after the intraperitoneal administration of dextrin 2-sulphate in patients with AIDS.

OBJECTIVE: To determine the safety and efficacy of the sulphated polysaccharide, dextrin 2-sulphate, when delivered to the lymphatic circulation by the peritoneal route. DESIGN: An open Phase I/II dose-escalation clinical study in which six patients with AIDS were treated with seven courses of dextrin 2-sulphate each lasting 1 month. METHODS: During each course of treatment, the drug was administered daily for 28 days using an intraperitoneal catheter. Viral load was measured at frequent intervals using a plasma tissue culture infectious dose (TCID) assay, a cellular TCID assay, p24 antigenaemia, HIV-1 RNA and HIV-1 DNA. Plasma beta-chemokine levels were also measured. RESULTS: Dose escalation was completed without toxicity. A total of 7 patient-months of treatment were completed. With increasing doses of dextrin 2-sulphate, the infectious plasma viraemia, cellular viraemia and p24 antigenaemia all fell during the period of drug administration, but with no significant change in HIV-1 RNA. This was associated with increased plasma levels of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. Dextrin 2-sulphate accumulated in peritoneal macrophages and induced the release of MIP-1alpha and MIP-1beta from these cells in vitro. These beta-chemokines could have augmented the cell surface-mediated anti-HIV-1 effect of dextrin 2-sulphate in vivo by binding to and blocking the CC-chemokine receptor-5. A second fall in infectious plasma viraemia, cellular viraemia, p24 antigenaemia and HIV-1 RNA was seen at day 100 which was then sustained for several months. A clinical improvement in Kaposi's sarcoma was also seen. CONCLUSIONS: Our results suggest that the intraperitoneal administration of dextrin 2-sulphate can reduce the replication of HIV-1 in patients with AIDS. With increasing doses of dextrin 2-sulphate, the fall in viral load was seen during the period of drug administration and again 2 months after completing treatment.

Evidence for a post-entry barrier to R5 HIV-1 infection of CD4 memory T cells.

BACKGROUND: HIV-1 strains R5 and X4 can infect CD4 memory T cells in vivo. Anti-CD3/28 stimulation induces beta-chemokines and CCR5 down-regulation and renders these cells resistant to R5 HIV-1 infection. Here we describe an additional cellular mechanism that blocks productive R5 HIV-1 infection of CD4 memory T cells. METHODS: Blood-derived CD4 memory T cells and CD4 T-cell clones were infected with primary R5 and X4 HIV-1 strains. Virus replication was correlated with CCR5 expression and beta-chemokine production. Virus entry and infectivity were measured by PCR for early and late products of HIV reverse transcription respectively. RESULTS: R5 strains were up to 1000-fold less infectious than X4 viruses for CD4 memory T cells. This resistance was independent of CCR5 levels and of the Delta-32 mutation and the CCR2-V64I/CCR5-59653T linked mutations. Blocking endogenous beta-chemokines relieved minimally this restriction. At the single cell level, CD4 memory cells were either permissive or non-permissive for R5 HIV-1 infection. R5 HIV titre was up to 10-fold lower than X4 virus titre even in a permissive clone. However, R5 viruses replicated as efficiently as X4 viruses in the permissive clone when neutralizing anti-beta chemokine antibodies were added. Non-permissive cells blocked a post-entry step of the virus life-cycle and expressed early but not late HIV transcripts. Neutralizing anti-beta chemokine antibodies promoted R5 virus replication marginally in the non-permissive clone. CONCLUSION: Some blood memory CD4 T cells retard R5 HIV-1 replication via endogenous beta-chemokines whereas others block productive R5 HIV-1 infection by an additional mechanism that interferes with a post-entry step of the virus life cycle. These natural barriers might contribute to lower pathogenicity of R5 HIV-1 strains for CD4 memory T cells than X4 viruses that emerge late in disease.

Reliable and reproducible LightCycler qPCR for HIV-1 DNA 2-LTR circles.

Highly active anti-retroviral therapy (HAART) has reduced the plasma load of HIV-1 to undetectable levels. It has however failed to eliminate the virus from other body compartments. Current methods for monitoring persistent viral replication in HIV-1+ patients require a large amount of blood and/or repeated tissue biopsies. Furthermore, some of the viral reservoirs, such as brain and eye, are inaccessible for sampling. The detection of episomal HIV-1 DNA 2-LTR circles in CD4+ cells is indicative of recent, acute infection events. This paper describes a reliable and reproducible LightCycler-based assay for the quantitative measurement of HIV-1 DNA 2-LTR circles in human peripheral blood mononuclear (PBMN) cells. It details the modifications to the DNA extraction procedure and to the LightCycler PCR procedure that were required to achieve this. This new surrogate marker of persistent viral replication can now be reliably, reproducibly and robustly used to study the clinical progress of large numbers of patients whose plasma HIV-1 RNA has been reduced to undetectable levels by anti-retroviral drugs.

LightCycler qPCR optimisation for low copy number target DNA.

The LightCycler is a rapid air-heated thermal cycler which incorporates a fluorimeter for the detection and quantification of Polymerase Chain Reaction (PCR) amplified products. It provides real-time cycle-by-cycle analysis of product generation. Amplification occurs in glass capillary tubes. The products are detected using a fluorescent double stranded DNA binding dye or fluorescent probes. However, conditions that work well in conventional PCR reactions do not readily translate to the LightCycler. Whilst using this new technology to study an infectious pathogen in human tissue samples, several parameters were identified which can have an adverse effect on the reliable and reproducible quantification of low copy number target DNA. They included abstraction of PCR reagents on glass, primer-dimer formation, non-specific product generation, and a failure to amplify low copy number target when it is present in a high background of human chromosomal DNA. For each problem identified, several solutions are described. Novel approaches are also described to ensure that amplification of target DNA and of the quantification standards occurs with the same efficiency. With appropriate changes to the protocols currently in use, LightCycler quantitative Polymerase Chain Reaction (LC-qPCR) can be used to achieve a level of accuracy that exceeds that of an enzyme immunoassay. The LC-qPCR optimisation strategies described are of particular relevance when applying this technology to the study of pathogens in tissue samples. The technique offers the enormous potential for reliable and reproducible quantitative PCR of low copy number target DNA.

Proteolytic processing of the Ada protein that repairs DNA O6-methylguanine residues in E. coli.

In extracts of E. coli treated with an adapting regime of MNNG, the induced 39kd Ada protein having O6-MeG-DNA methyltransferase activity is processed to a 19kd active domain corresponding to the C-terminal half of the intact protein. This proteolytic processing has been followed on Western immunoblots using antisera raised against the 19kd fragment. Initial processing at 25 degrees C or 37 degrees C mainly generates a fragment of mol. wt. 24kd which then undergoes a slower second cleavage to generate the 19kd active domain. Preceding this second cleavage site is a sequence of amino acids Thr- -Gly-Met-Thr- -Lys that also occurs at another site in the N-terminal half of the 39kd methyltransferase. It is proposed that this sequence is a recognition site for proteolytic activity. On the basis of cleavage of the Ada protein at either one or both of these sites, fragments may be generated of mol. wt. 24kd and 19kd containing the active site for O6-methylguanine and O4-methylthymine repair, and 15kd and 20kd, containing the active site for methylphosphotriester repair. These observations explain previous reports by others on the existence in cell extracts of multiple methyltransferase activities of different sizes recognizing O-methyl lesions in DNA. The cellular protease involved is resistant to a wide range of protease inhibitors.

Multiple hypersensitivity to mutagens in a cell strain (46BR) derived from a patient with immuno-deficiencies.

46BR is a fibroblast cell strain established from an individual with hypogammaglobulinaemia. The cells are unique in showing hypersensitivity to the lethal effects of a wide range of DNA-damaging agents. Thus they are hypersensitive to gamma- and 254-nm UV-irradiation and show a limited capacity to repair potentially lethal gamma-irradiation damage when compared with fibroblasts from normal individuals. A slight hypersensitivity to mitomycin C was also revealed but we were not able to discriminate 46BR from normals with 4-nitroquinoline oxide. The cells were hypersensitive to the alkylating agents, dimethyl sulphate, methyl methanesulphonate, ethyl methanesulphonate, N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea but not N-ethyl-N-nitrosourea. A consideration of the spectra of DNA lesions produced by these alkylating agents together with the sensitivity to ionising radiation and mitomycin C suggests that 46BR cells are defective in a repair step that is common to all agents. We suggest that the cells are defective in DNA polymerisation or ligation. Support for this suggestion comes from the absence of any hypersensitivity to N-ethyl-N-nitrosourea since its major reaction products are not removed by excision pathways that require polymerisation and ligation.

Patients' perspective of wide-awake hand surgery--100 consecutive cases.

The wide-awake hand surgery (WAHS) technique involves injecting lidocaine with adrenaline for hand surgical procedures that are done without the use of tourniquets, sedation, regional or general anaesthetic. This is a retrospective review of the first 100 consecutive patients who underwent operations using this technique at our centre. The operations included carpal and cubital tunnel decompression, trapeziectomy, tendon transfer, and tenolysis. A questionnaire adapted from Lalonde's previous work on wide-awake surgery was used to assess patients' experiences. Sixty-five percent of the patients responded to the postal questionnaire, the majority reporting a high satisfaction level. Ninety-one percent of responders reported that the operation was less painful or comparable with a procedure at the dentist; 86% would prefer to be wide-awake if they needed to have hand surgery again, and 90% stated they would recommend WAHS to a friend.

Identification and analysis of the transport/capsid assembly protein (tp/cap) gene of human herpesvirus-6 (HHV6).

The transport/capsid assembly protein (tp/cap) gene of human herpesvirus 6 (HHV6) strain U1102 has been identified and localized on the restriction enzyme map of the viral genome, to the EcoRI-Q fragment. The complete DNA sequence of the tp/cap gene was determined. The tp/cap gene encodes a protein product of 726 amino acids and has the strongest similarity with the homologous gene (HCMV UL56) from HCMV. Upstream of the tp/cap open reading frame is the gene for the major DNA binding protein (mdbp) and downstream is the glycoprotein B (gB) gene. This gene block arrangement is common to all herpesviruses.

Polymerase chain reaction in situ: an appraisal of an emerging technique.

Polymerase chain reaction (PCR) in situ is a new technique which promises to enhance considerably our ability to detect a few copies of target nucleic acid sequences in fixed tissues and cells. It has an enormous potential for application in diagnostic histopathology of viral diseases and in the study of gene expression. PCR in situ is, however, technically difficult, and amplification of the target DNA is only 30-300 fold. In this article we present an overview of PCR in situ techniques used to amplify both DNA and RNA targets (RT-PCR in situ). We also identify problems which can reduce the efficiency of the technique or which can give rise to false-positive results. They include (1) the inhibitory effects of cross-linking of histones to DNA or PCR amplification, (2) abstraction of PCR reagents by tissue-bonding agents which are used to coat glass slides, (3) poor denaturation of target DNA and subsequent DNA renaturation due to extensive cross-linking of histones to DNA, or because of incorrect temperature regulation of thermal cyclers, (4) false-positive results which arise from end-labelling of DNA strand breaks by Taq polymerase, and (5) diffusion of PCR products into and out of cells leading to false-positive results. We present some of the approaches that have been used to overcome some of these difficulties and suggest new avenues for investigation to improve this technique further.