Palliative medicine in the intensive care unit: needs, delivery, quality.

BACKGROUND: 15%-20% of critical care patients die during their hospital admission. This service evaluation assesses quality of palliative care in intensive care units (ICUs) compared with national standards. METHODS: Retrospective review of records for all patients who died in four ICUs (irrespective of treatment limitation) between 1 June and 31 July 2019. Descriptive statistics reported for patient characteristics, length of stay, admission route, identification triggers and palliative care delivery. RESULTS: Forty-five patients died, two records were untraced, thus N=43. The dying process was recognised in 88% (n=38). Among those where dying was recognised (N=35), 97% (34) had documented family discussion before death, 9% (3) were offered religious/spiritual support, 11% (4) had review of hydration/nutrition and 6% (2) had documented preferred place of death. Prescription of symptom control medications was complete in 71% (25) opioids, 34% (12) haloperidol, 54% (19) midazolam and 43% (15) hyoscine. Combining five triggers-length of stay >10 days prior to ICU admission 7% (3), multiorgan failure ≥3 systems 33% (14), stage IV malignancy 5% (2), post-cardiac arrest 23% (10) and intracerebral haemorrhage requiring mechanical ventilation 12% (5)-identified 60% (26) of patients. Referral to the palliative care team was seen in 14% (5), and 8% (3) had specialist palliative care team review. CONCLUSIONS: Recognition of dying was high but occurred close to death. Family discussions were frequent, but religious/spiritual needs, hydration/nutrition and anticipatory medications were less often considered. The ICUs delivered their own palliative care in conjunction with specialist palliative care input. Combining five triggers could increase identification of palliative care needs, but a larger study is needed.

Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sjogren's syndrome.

Sjogren's syndrome (SjS) patients often have a variety of extraglandular manifestations including neurological and gastrointestinal involvement. In this study we evaluated the diagnostic performance of luciferase immunoprecipitation system (LIPS) that employs mammalian cell-produced recombinant antigens for analyzing SjS autoantibody responses. LIPS testing of mammalian cell-produced La, Ro60 and Ro52 recombinant antigens with defined commercial antibodies demonstrated highly specific immunoprecitation of each antigen without cross-reactivity. Next, sera from 57 SjS and 25 volunteers were evaluated by LIPS against a panel of human autoantigens. LIPS detected robust anti-La antibody levels in 43/57 SjS patients (75% sensitivity) and markedly outperformed an ELISA (46% sensitivity). Profiling of other SjS-associated autoantigens revealed the presence of anti-Ro60, anti-Ro52 in 63% and 61%, of SjS patients, respectively. Interestingly, a C-terminal fragment of Ro52 (Ro52-Delta2), a protein fragment not previously found to be antigenic by ELISA, also showed positive immunoreactivity in 42/57 SjS patients (65% sensitivity). Additional profiling of other autoantigens revealed that certain SjS patients also showed positive immunoreactivity with thyroid peroxidase (14%), AQP-4 (12%) and the H(+)/K(+) gastric ATPase (16%) suggesting potential autoantibody attack of thyroid, neuronal and gastric parietal cells, respectively. These heterogeneous autoantibody responses detected by LIPS in SjS will likely be useful for diagnosis and for evaluating extraglandular manifestations.

A four-antigen mixture for rapid assessment of Onchocerca volvulus infection.

BACKGROUND: Onchocerciasis, an infection caused by the filarial nematode Onchocerca volvulus, is a major public health concern. Given the debilitating symptoms associated with onchocerciasis and concerns about recrudescence in areas of previous onchocerciasis control, more efficient tools are needed for diagnosis and monitoring of control measures. We investigated whether luciferase immunoprecipitation systems (LIPS) may be used as a more rapid, specific, and standardized diagnostic assay for Onchocerca volvulus infection. METHODS: Four recombinantly produced Onchocerca volvulus antigens (Ov-FAR-1, Ov-API-1, Ov-MSA-1 and Ov-CPI-1) were tested by LIPS on a large cohort of blinded sera comprised of both uninfected controls and patients with a proven parasitic infection including Onchocerca volvulus (Ov), Wuchereria bancrofti (Wb), Loa loa (Ll), Strongyloides stercoralis (Ss), and with other potentially cross-reactive infections. In addition to testing all four Ov antigens separately, a mixture that tested all four antigens simultaneously was evaluated in the standard 2-hour incubation format as well as in a 15-minute rapid LIPS format. FINDINGS: Antibody responses to the four different Ov antigens allowed for unequivocal differentiation between Ov-infected and uninfected control sera with 100% sensitivity and 100% specificity. Analysis of the antibody titers to each of these four antigens in individual Ov-infected sera revealed that they were markedly different and did not correlate (r(S) = -0.11 to 0.58; P = 0.001 to 0.89) to each other. Compared to Ov-infected sera, patients infected with Wb, Ll, Ss, and other conditions had markedly lower geometric mean antibody titers to each of the Ov 4 antigens (P<0.0002 for each antigen). The simplified method of using a mixture of the 4 Ov antigens simultaneously in the standard format or a quick 15-minute format (QLIPS) showed 100% sensitivity and 100% specificity in distinguishing the Ov-infected sera from the uninfected control sera. Finally, the QLIPS format had the best performance with 100% sensitivity and specificity values of 76%, 84% and 93% for distinguishing Ov from Wb, Ll and Ss-infected sera. CONCLUSIONS: The multi-antigen LIPS assay can be used as a rapid, high throughput, and specific tool to not only to diagnose individual Ov infections but also as a sensitive and potentially point-of-care method for early detection of recrudescent infections in areas under control and for mapping new areas of transmission of Ov infection.

Four-antigen mixture containing v-cyclin for serological screening of human herpesvirus 8 infection.

Improved diagnostic reagents and testing are currently needed for the serological detection of human herpesvirus 8 (HHV-8) infections. We evaluated the luciferase immunoprecipitation systems (LIPS) for profiling antibody responses to a panel of HHV-8 proteins for diagnosis of Kaposi sarcoma (KS)-infected individuals. Using a pilot serum set, LIPS detected robust antibody responses to several known antigens, and a screen of 14 additional HHV-8 proteins identified v-cyclin as a potentially new diagnostic antigen. In evaluating a training-serum set, a four-antigen panel (K8.1, v-cyclin, ORF65, and a LANA fragment) was found to provide sufficient information for diagnosis. Analysis of a validation serum set using the combined results from these four separate antigen tests showed 100% sensitivity and 100% specificity. Furthermore, a LIPS format using a mixture of the four antigens, which simplifies data collection and analysis, closely matched the diagnostic performance of the combined separate tests (R = 0.95). This four-antigen mixture format analyzed with the validation serum set also showed 100% sensitivity and 100% specificity but was not statistically different from two separate enzyme-linked immunosorbent assays (94% sensitivity and 100% specificity) using baculovirus-produced LANA and bacterially produced K8.1. Heat map analysis of KS patient antibody titers revealed marked heterogeneity in humoral responses to this four-antigen panel. Overall, the LIPS assay showed 97% sensitivity, and positive anti-v-cyclin antibodies were detected in approximately 75% of the KS sera. These results suggest that LIPS screening using an antigen mixture is a sensitive and high-throughput method for serological screening of HHV-8 infection in individuals with KS.

Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay.

Highly quantitative and high-throughput serological tests for evaluation of humoral responses to herpes simplex virus 1 (HSV-1) and HSV-2 are not available. The efficacy of luciferase immunoprecipitation system (LIPS) assays for antibody profiling and serologic diagnosis of HSV-1 and HSV-2 infection was investigated using a panel of five recombinant HSV antigens. Plasma samples from subjects seropositive for HSV-1 and/or HSV-2 or seronegative for HSV-1 and HSV-2 that had previously been analyzed by Western blotting and the Focus Plexus immunoassay were evaluated. The LIPS test measuring anti-gG1 antibody titers was 96% sensitive and 96% specific for detecting HSV-1 infection, compared with the Focus immunoassay, and was 92% sensitive and 96% specific, compared with Western blotting. The results for the anti-gG2 LIPS test for HSV-2 precisely matched those for Western blotting, with 100% sensitivity and 100% specificity, and showed robust antibody titers in all the HSV-2-infected samples that were over 1,000 times higher than those in HSV-2-negative or HSV-1-positive samples. Antibodies to three additional HSV-2 proteins, gB, gD, and ICP8, were detected in many of the HSV-1- and/or HSV-2-infected plasma samples and showed preferentially higher immunoreactivity in HSV-2-infected plasma. The titers of antibodies to these three HSV-2 antigens also significantly correlated with each other (R=0.75 to 0.81; P<0.0001). These studies indicate that the robust anti-gG1 and anti-gG2 antibody responses detected by LIPS assays are useful for HSV-1 and HSV-2 detection and suggest that profiling of antibody responses to a panel of HSV proteins may be useful for characterizing individual humoral responses to infection and for monitoring responses to vaccines.

Anti-HTLV antibody profiling reveals an antibody signature for HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP).

BACKGROUND: HTLV-I is the causal agent of adult T cell leukemia (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Biomarkers are needed to diagnose and/or predict patients who are at risk for HAM/TSP or ATLL. Therefore, we investigated using luciferase immunoprecipitation technology (LIPS) antibody responses to seven HTLV-I proteins in non-infected controls, asymptomatic HTLV-I-carriers, ATLL and HAM/TSP sera samples. Antibody profiles were correlated with viral load and examined in longitudinal samples. RESULTS: Anti-GAG antibody titers detected by LIPS differentiated HTLV-infected subjects from uninfected controls with 100% sensitivity and 100% specificity, but did not differ between HTLV-I infected subgroups. However, anti-Env antibody titers were over 4-fold higher in HAM/TSP compared to both asymptomatic HTLV-I (P < 0.0001) and ATLL patients (P < 0.0005). Anti-Env antibody titers above 100,000 LU had 75% positive predictive value and 79% negative predictive value for identifying the HAM/TSP sub-type. Anti-Tax antibody titers were also higher (P < 0.0005) in the HAM/TSP compared to the asymptomatic HTLV-I carriers. Proviral load correlated with anti-Env antibodies in asymptomatic carriers (R = 0.76), but not in HAM/TSP. CONCLUSION: These studies indicate that anti-HTLV-I antibody responses detected by LIPS are useful for diagnosis and suggest that elevated anti-Env antibodies are a common feature found in HAM/TSP patients.

A new luminescence assay for autoantibodies to mammalian cell-prepared insulinoma-associated protein 2.

OBJECTIVE: Insulinoma-associated protein 2 (IA-2) is a major autoantigen in type 1 diabetes, and IA-2 autoantibodies are routinely detected by a liquid-phase radioimmunoprecipitation assay. The present experiments were initiated to develop a new assay that does not require the use of radioisotopes or autoantigens prepared in bacteria or by in vitro transcription/translation. RESEARCH DESIGN AND METHODS: IA-2 luciferase fusion protein was expressed in mammalian cells and assayed for autoantibodies by liquid-phase luciferase immunoprecipitation. RESULTS: Our study showed that there was no significant difference between the luciferase immunoprecipitation and the radioimmunoprecipitation assays in sensitivity and specificity, and comparison of the two assays revealed a high correlation coefficient (R(2) = 0.805). CONCLUSIONS: The luciferase system offers a robust, inexpensive, nonradioactive method for the detection of autoantibodies to mammalian cell-prepared IA-2 and could be of practical value at the clinical level.

Modulation of Kv4.2 K+ currents by neuronal interleukin-16, a PDZ domain-containing protein expressed in the hippocampus and cerebellum.

Neuronal interleukin-16 (NIL-16) is a multi-PDZ domain protein expressed in post-mitotic neurons of the hippocampus and cerebellum. NIL-16 contains four PDZ domains, two of which are located within the neuron-specific N-terminal region. In yeast two-hybrid systems, the N-terminus of NIL-16 interacts with several ion channel proteins, including the Kv4.2 subunit of A-type K(+) channels. Here we provide evidence that NIL-16, through interactions with Kv4.2, influences Kv4.2 channel function and subcellular distribution. Specifically, coexpression of NIL-16 with Kv4.2 in COS-7 cells results in a significant reduction in whole-cell A-type current densities; however, when the Kv4.2 PDZ-ligand domain is mutated, Kv4.2 current densities are not affected by NIL-16 coexpression. Moreover, single-channel conductance was not influenced by the presence of NIL-16. In hippocampal neurons, A-type current densities are increased by conditions that inhibit interactions between NIL-16 and Kv4.2, such as overexpression of the Kv4.2 C-terminal PDZ-ligand domain and treatment with small-interfering RNA duplexes that reduce NIL-16 expression. Results of surface biotinylation assays using COS-7 cells suggest that Kv4.2 surface expression levels are reduced by coexpression with NIL-16. In addition, coexpression of NIL-16 with Kv4.2 induces Kv4.2 to form dense intracellular clusters; whereas without NIL-16, Kv4.2 channels cells are dispersed. Taken together, these data suggest that interactions between Kv4.2 and NIL-16 may reduce the number of functional Kv4.2-containing channels on the cell surface. In summary, NIL-16 may provide a novel form of A-type K(+) channel modulation that is localized specifically to neurons of the hippocampus and cerebellum.

The vacuolar-ATPase inhibitor bafilomycin and mutant VPS35 inhibit canonical Wnt signaling.

Endosomal acidification and transport are essential functions in signal transduction. Recent data suggest that Wnt signaling requires intact endosomal transport machinery but the effects of endosomal acidification on Wnt signal transduction have not been evaluated. Here we report that bafilomycin, a specific inhibitor of the vacuolar proton ATPase that blocks endosomal acidification, inhibits canonical Wnt signal transduction initiated by Wnt ligand and partially inhibits signaling initiated by disheveled. Bafilomycin does not affect Tcf promoter activation by beta-catenin. These data indicate that endosomal acidification is necessary for Wnt signaling. To identify interactions between endosomal transport proteins and Wnt receptors, we performed a GST fusion protein pulldown experiment and identified a possible indirect interaction between the LRP6 intracellular domain and vacuolar protein sorting protein 35 (VPS35). We show that an N-terminal deletion mutant of VPS35 reduces canonical Wnt signaling in HEK-293 cells expressing exogenous Wnt-1. These data suggest that endosomal V-type ATPase activity and retromer trafficking proteins are functionally important in Wnt signal transduction.