Are positive-pressure ventilation lobby rooms effective for protective and source isolation?

High-efficiency particulate air supplied to a positive-pressure ventilation lobby (PPVL) in isolation rooms offers the dual advantage of protective and source isolation. This study demonstrates the in-use validity of PPVL rooms for protective isolation of patients. Of the 48 PPVL air samples investigated, Aspergillus fumigatus was detected from only one (2%) sample. Local and remote monitoring of the PPVL rooms is essential for the safety of patients and healthcare workers. Remote and point-of-use engineering controls are essential for ongoing ventilation monitoring, but this should be complemented by visual inspection of the isolation suite. Periodic microbiological monitoring should also be considered with other control measures.

Antifungal stewardship in critical care: Implementing a diagnostics-driven care pathway in the management of invasive candidiasis.

BACKGROUND: Invasive candidiasis (IC) is the most common invasive fungal disease in patients admitted to critical care and is associated with high mortality rates. Diagnosis can be delayed by the poor sensitivity of culture-based methods, leading to unnecessary use of empirical antifungal therapy (EAFT). The fungal biomarker (1-3)-ß-d-glucan (BDG) has been shown to aid in the diagnosis of IC in critical care and has been incorporated into antifungal stewardship (AFS) programmes. AIM: To describe our experience using a diagnostics-driven AFS programme incorporating the fungal biomarker BDG, analyse its impact on antifungal therapy (AFT), and gain an improved understanding of the epidemiology of IC in our critical care unit (CrCU). METHODS: An AFS care pathway incorporating BDG was introduced in the CrCU in St James's Hospital, Dublin. Following an educational programme, compliance with the pathway was prospectively audited between December 1st, 2017 and July 31st, 2018. RESULTS AND CONCLUSION: One hundred and nine AFT episodes were included, of which 95 (87%) had a BDG sent. Of those with BDG results available at the time of decision-making, 38 (63%) were managed in accordance with the care pathway. In compliant episodes without IC, median EAFT duration was 5.5 days [IQR 4-7] and no increase in mortality or subsequent IC was observed. Although adopting a diagnostics-driven approach was found to be useful in the cohort of patients with BDG results available, the use of once-weekly BDG testing did not result in an observed reduction in the consumption of anidulafungin, highlighting an important limitation of this approach.

Prevention of healthcare-associated invasive aspergillosis during hospital construction/renovation works.

The association between healthcare-associated invasive aspergillosis and hospital construction/building works is well recognized. This infection can cause significant morbidity and mortality and imposes a substantial burden on the healthcare system. The population of patients at risk for this opportunistic infection has expanded and multi-triazole drug resistance has emerged globally. Hence the need for a multi-faceted approach to prevent acquisition of invasive aspergillosis in acute care settings. This article is a summary of the Irish National Guidelines for the prevention of healthcare-associated aspergillosis which is based on published reports, international clinical guidelines, official engineering standards, and technical guidelines. We discuss the key recommendations and strategies for the prevention of invasive aspergillosis from the planning/pre-construction, construction, and post-construction phases. The importance of multi-disciplinary team involvement, education, and communication is emphasized.

A Unique Case of Concurrent Long Bone Osteomyelitis and Empyema Caused by Fusobacterium nucleatum.

We describe a patient presenting with constitutional symptoms, poor oral hygiene and leg swelling who was diagnosed with Fusobacterium nucleatum osteomyelitis of femur and empyema. Long bone osteomyelitis is rarely caused by this microorganism. This unusual case was successfully managed with drainage and antimicrobial therapy.

Reduced pro-inflammatory responses to Staphylococcus aureus bloodstream infection and low prevalence of enterotoxin genes in isolates from patients on haemodialysis.

Patients with end-stage renal failure undergo regular haemodialysis (HD) and often develop episodes of Staphylococcus aureus bloodstream infection (BSI), which can re-occur. However, clinically, patients on HD, with S. aureus BSI, respond well to treatment, rarely developing overt signs of sepsis. We investigated the contributions of bacterial virulence and cytokine responses to the clinical course of S. aureus BSI in HD and non-HD patients. Seventy patients were recruited, including 27 (38.6 %) patients on HD. Isolates were spa-typed and virulence and antimicrobial resistance gene carriage was investigated using DNA microarray analysis. Four inflammatory cytokines, IL-6, RANTES, GROγ and leptin, were measured in patient plasma on the day of diagnosis and after 7 days. There was no significant difference in the prevalence of genotypes or antimicrobial resistance genes in S. aureus isolates from HD compared to non-HD patients. The enterotoxin gene cluster (containing staphylococcal enterotoxins seg, sei, sem, sen, seo and seu) was significantly less prevalent among BSI isolates from HD patients compared to non-HD patients. Comparing inflammatory cytokine response to S. aureus BSI in HD patients to non-HD patients, IL-6 and GROγ were significantly lower (p = 0.021 and p = 0.001, respectively) in HD patients compared to other patients on the day of diagnosis and RANTES levels were significantly lower (p = 0.025) in HD patients on day 7 following diagnosis. Lowered cytokine responses in HD patients and a reduced potential for super-antigen production by infecting isolates may partly explain the favourable clinical responses to episodes of S. aureus BSI in HD patients that we noted clinically.

A tacrolimus-related immunosuppressant with reduced toxicity.

BACKGROUND: Tacrolimus (FK506) has potent immunosuppressive properties reflecting its ability to block the transcription of lymphokine genes in activated T cells through formation of a complex with FK506 binding protein-12, which inhibits the phosphatase activity of calcineurin. The clinical usefulness of tacrolimus is limited, however, by severe adverse effects, including neurotoxicity and nephrotoxicity. Although this toxicity, like immunosuppression, appears mechanistically related to the calcineurin inhibitory action of the drug, a large chemistry effort has been devoted to search for tacrolimus analogs with reduced toxicity but preserved immunosuppressive activity that might have enhanced therapeutic utility. METHODS: Here, we report on the identification of such an analog, which was synthetically derived from ascomycin (ASC), the C21 ethyl analog of tacrolimus, by introducing an indole group at the C32 position. The profile of biological activity of indolyl-ASC was characterized in rodent models of immunosuppression and toxicity. RESULTS: Indolyl-ASC was found to exhibit an immunosuppressive potency equivalent to that of tacrolimus in T-cell activation in vitro and in murine transplant models, even though indolyl-ASC bound about 10 times less to intracellular FK506 binding protein-12 than tacrolimus or ASC. Further evaluation of indolyl-ASC revealed that it is threefold less potent than tacrolimus in inducing hypothermia, a response that may reflect neurotoxicity, and in causing gastrointestinal transit alterations in mice. Moreover, indolyl-ASC was at least twofold less nephrotoxic than tacrolimus upon 3-week oral treatment in rats. CONCLUSIONS: Altogether, these data indicate a modest but definite improvement in the therapeutic index for indolyl-ASC compared with tacrolimus in rodent models.

Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.

The voltage activated K+ channel (Kv1.3) has recently been identified as the molecule that sets the resting membrane potential of peripheral human T lymphoid cells. In vitro studies indicate that blockage of Kv1.3 inhibits T cell activation, suggesting that Kv1.3 may be a target for immunosuppression. However, despite the in vitro evidence, there has been no in vivo demonstration that blockade of Kv1.3 will attenuate an immune response. The difficulty is due to species differences, as the channel does not set the membrane potential in rodent peripheral T cells. In this study, we show that the channel is present on peripheral T cells of miniswine. Using the peptidyl Kv1.3 inhibitor, margatoxin, we demonstrate that Kv1.3 also regulates the resting membrane potential, and that blockade of Kv1.3 inhibits, in vivo, both a delayed-type hypersensitivity reaction and an Ab response to an allogeneic challenge. In addition, prolonged Kv1.3 blockade causes reduced thymic cellularity and inhibits the thymic development of T cell subsets. These results provide in vivo evidence that Kv1.3 is a novel target for immunomodulation.

Association of serine protease with the rise of intracellular calcium in cytotoxic T lymphocytes.

The precise role of the granular enzyme A (granzyme A), a serine protease, in the lytic process of cytotoxic T lymphocytes (CTL) is not clear. We have recently constructed a CTL line transfected with the antisense gene of granzyme A (a-GrA). These a-GrA CTL had lower GrA activity as well as decreased lytic activities, as measured by 51Cr and by DNA degradation assays. Furthermore, at low effector:target ratio (1:8) in prolonged lytic assays, they could not lyse targets as rapidly as the control CTL. When we examined their ability to exocytose BLT (CBZ-L-lys-thiobenzyl)-esterase in the presence of anti-CD3 antibody, the a-GrA CTL exocytosed poorly compared to the parental CTL or control transfectant with a CAT gene. Most strikingly, a-GrA cells could not release intracellular stores of Ca2+ in response to anti-CD3 induction, although the Ca2+ flux was normal when they were stimulated with ionomycin. When the parental CTL was treated with a specific benzyllactam inhibitor of BLT-esterase or N-tosyl-L-phenylalanylchloromethyl ketone, the Ca2+ flux induced by anti-CD3 was also suppressed. We propose that granzyme A is involved in the signal transduction pathway that causes the rise of the intracellular calcium.

CTL lysis: there is a hyperbolic relation of killing rate to exocytosable granzyme A for highly cytotoxic murine cytotoxic T lymphocytes.

The lysis of susceptible targets by efficient cytotoxic T lymphocytes (CTL) increases both with time and with the ratio of CTL to target. Simple methods for calculating a killing rate constant from the time dependence of killing and for calculating the relation of the killing rate constant to the concentration of exocytosable granzyme A are given. Application of these methods to the killing of target cells by the highly efficient mouse CTL AR1 is presented. AR1 needed granzyme A for efficient killing. AR1 contained sufficient exocytosable granzyme A to kill at about 80% of the rate possible at infinite exocytosable granzyme A.

Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation.

The role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca(2+)-activated potassium channels (PK,Ca) and voltage-gated potassium channels (PK,V) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (IL-2 and interferon gamma) are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,Ca and PK,V, we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for PK,V. These studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca(2+)-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

A single administration of LFA-1 antibody confers prolonged allograft survival.

C57BL/6 (B6) thyroid gland transplanted to the left kidney capsule of an allogeneic (BALB/c) host was typically rejected in 14 days. A single administration of 500 micrograms of an antibody to the adhesion molecule, leucocyte function-associated antigen (LFA-1, CD11a), prevented all thyroid allograft rejection for at least 70 days. Fifty percent of the treated recipients retained intact allografts for 470 days. However, the same treatment with anti-CD11a could not protect a sensitized BALB/c mouse from rejecting a second B6 thyroid allograft. Production of donor-specific alloantibodies elicited by allograft rejection was also inhibited in this system. In this transplant model, the Ab therapy is more efficacious than that of FK506, administered daily for 14 days at 15 mg/kg. These results demonstrate the remarkable effect of an anti-LFA-1 antibody in promotion of allograft survival.

Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.

Ovarian teratocarcinomas in LT/Sv mice carrying t-mutations.

Ovarian teratomas that result from parthenogenetic activation of oocytes provide a double tool for developmental genetics. First, they provide a way of measuring recombination between a gene and its centromere. Second, in the absence of crossing over there is the potential of producing tumors that are homozygous for genes that would be lethal in the course of in utero embryonic development. We have applied both aspects to several t-haplotypes containing different early acting t-lethal genes. In a study of 26 tumors, genotyped by Southern blot analysis of the major histocompatibility complex (MHC), we measured the distance between the centromere and the start of the t-complex as 5.6 +/- 2.3 cM. We found a marked deficiency of t-homozygous genotypes among the tumors we studied, although T/T genotypes formed teratomas at levels comparable to controls. None of the lethal t-haplotypes we studied permit homozygous embryos to develop to the primitive streak stage, while T/T embryos do develop essentially normally through that stage. Thus, although the total number of tumors observed from t-bearing mice was small, the great difference in the incidence of t/t tumors versus the incidence of T/T tumors suggests strongly that the parthenogenetic embryos that convert to teratocarcinomas must first pass through some of the stages of normal early development, including the formation of three germ layers and the primitive streak.

Analysis of major histocompatibility complex haplotypes of t-chromosomes reveals that the majority of diversity is generated by recombination.

t-chromosomes are natural polymorphisms in feral populations of mice that are thought to be descended from a single ancestral chromosome. They carry an inversion of at least 10 cM surrounding the major histocompatibility complex (MHC) that effectively prevents recombination between a t-bearing chromosome and wild type chromosomes. However, on the rare occasion when two different t-chromosomes meet in a wild female, recombination occurs at an apparently normal rate. Since they contain the highly polymorphic MHC, their limited origin and restricted chances for recombination make t-chromosomes a valuable tool for studying the relative contributions of mutation and recombination to the generation of diversity. Using 13 different serological reagents to class I antigens, and studying restriction enzyme polymorphisms detected with three molecular probes for class II genes examined with three endonucleases, we present data indicating that the major factor responsible for the diversity of class I antigens is recombination, but that for class II genes, mutation must play an important role in addition to recombination.