Risk of neurosyphilis in HIV-infected persons with syphilis lacking signs or symptoms of central nervous system infection.

OBJECTIVES: People living with HIV (PLWH) are at increased risk of asymptomatic neurosyphilis; thus, it has been common practice to perform a lumbar puncture (LP) in all PLWH presenting with syphilis regardless of stage, signs or symptoms. However, this practice varies widely among clinicians. Our objective was to elucidate the number of LPs required to diagnose a single case of asymptomatic neurosyphilis. METHODS: We performed an electronic health record (EHR) review of PLWH who were diagnosed with syphilis of any stage over a 10-year period. EHRs were reviewed to determine the number of subjects who had an LP performed, what proportion had neurological signs or symptoms, and whether a diagnosis of neurosyphilis was made at presentation or follow-up. RESULTS: In 261 separate episodes of syphilis in 230 subjects, we found the major risk factors for asymptomatic neurosyphilis to be low CD4 T-cell count (P = 0.0007), high rapid plasma reagin (RPR) titre (P = 0.019) and lack of HIV virological suppression (P = 0.003). The majority of our subjects (78%) with neurosyphilis presented with central nervous system (CNS) symptoms. We estimate, if standard practice is to perform LP in all patients, that the number needed to test (NNTT) = 38. CONCLUSIONS: This large number of potentially unnecessary LPs, along with heterogeneity of presentation, and the never-nil risk of asymptomatic neurosyphilis should be incorporated into clinical decision-making. The majority of PLWH presenting with a serological diagnosis of syphilis, but no neurological signs or symptoms, do not necessarily require an LP for an evaluation of asymptomatic neurosyphilis.

Automated symptom and treatment side effect monitoring for improved quality of life among adults with diabetic peripheral neuropathy in primary care: a pragmatic, cluster, randomized, controlled trial.

AIMS: To evaluate the effectiveness of automated symptom and side effect monitoring on quality of life among individuals with symptomatic diabetic peripheral neuropathy. METHODS: We conducted a pragmatic, cluster randomized controlled trial (July 2014 to July 2016) within a large healthcare system. We randomized 1834 primary care physicians and prospectively recruited from their lists 1270 individuals with neuropathy who were newly prescribed medications for their symptoms. Intervention participants received automated telephone-based symptom and side effect monitoring with physician feedback over 6 months. The control group received usual care plus three non-interactive diabetes educational calls. Our primary outcomes were quality of life (EQ-5D) and select symptoms (e.g. pain) measured 4-8 weeks after starting medication and again 8 months after baseline. Process outcomes included receiving a clinically effective dose and communication between individuals with neuropathy and their primary care provider over 12 months. Interviewers collecting outcome data were blinded to intervention assignment. RESULTS: Some 1252 participants completed the baseline measures [mean age (sd): 67 (11.7), 53% female, 57% white, 8% Asian, 13% black, 20% Hispanic]. In total, 1179 participants (93%) completed follow-up (619 control, 560 intervention). Quality of life scores (intervention: 0.658 ± 0.094; control: 0.653 ± 0.092) and symptom severity were similar at baseline. The intervention had no effect on primary [EQ-5D: -0.002 (95% CI -0.01, 0.01), P = 0.623; pain: 0.295 (-0.75, 1.34), P = 0.579; sleep disruption: 0.342 (-0.18, 0.86), P = 0.196; lower extremity functioning: -0.079 (-1.27, 1.11), P = 0.896; depression: -0.462 (-1.24, 0.32); P = 0.247] or process outcomes. CONCLUSIONS: Automated telephone monitoring and feedback alone were not effective at improving quality of life or symptoms for people with symptomatic diabetic peripheral neuropathy. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02056431).

Mapping sources of noise in an intensive care unit.

Excessive noise in hospitals adversely affects patients' sleep and recovery, causes stress and fatigue in staff and hampers communication. The World Health Organization suggests sound levels should be limited to 35 decibels. This is probably unachievable in intensive care units, but some reduction from current levels should be possible. A preliminary step would be to identify principal sources of noise. As part of a larger project investigating techniques to reduce environmental noise, we installed a microphone array system in one with four beds in an adult general intensive care unit. This continuously measured locations and sound pressure levels of noise sources. This report summarises results recorded over one year. Data were collected between 7 April 2017 and 16 April 2018 inclusive. Data for a whole day were available for 248 days. The sound location system revealed that the majority of loud sounds originated from extremely limited areas, very close to patients' ears. This proximity maximises the adverse effects of high environmental noise levels for patients. Some of this was likely to be appropriate communication between the patient, their caring staff and visitors. However, a significant proportion of loud sounds may originate from equipment alarms which are sited at the bedside. A redesign of the intensive care unit environment to move alarm sounds away from the bed-side might significantly reduce the environmental noise burden to patients.

Changes in health-related quality of life after discharge from an intensive care unit: a systematic review.

Quality of life after critical illness is becoming increasingly important as survival improves. Various measures have been used to study the quality of life of patients discharged from intensive care. We systematically reviewed validated measures of quality of life and their results. We searched PubMed, CENTRAL, CINAHL, Web of Science and Open Grey for studies of quality of life, measured after discharge from intensive care. We categorised studied populations as: general; restricted to level-3 care or critical care beyond 5 days; and septic patients. We included quality of life measured at any time after hospital discharge. We identified 48 studies. Thirty-one studies used the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36) and 19 used the EuroQol-5D (EQ-5D); eight used both and nine used alternative validated measures. Follow-up rates ranged from 26-100%. Quality of life after critical care was worse than for age- and sex-matched populations. Quality of life improved for one year after hospital discharge. The aspects of life that improved most were physical function, physical role, vitality and social function. However, these domains were also the least likely to recover to population norms as they were more profoundly affected by critical illness.

Antrodia cinnamomea reduces obesity and modulates the gut microbiota in high-fat diet-fed mice.

BACKGROUND: Obesity is associated with gut microbiota dysbiosis, disrupted intestinal barrier and chronic inflammation. Given the high and increasing prevalence of obesity worldwide, anti-obesity treatments that are safe, effective and widely available would be beneficial. We examined whether the medicinal mushroom Antrodia cinnamomea may reduce obesity in mice fed with a high-fat diet (HFD). METHODS: Male C57BL/6J mice were fed a HFD for 8 weeks to induce obesity and chronic inflammation. The mice were treated with a water extract of A. cinnamomea (WEAC), and body weight, fat accumulation, inflammation markers, insulin sensitivity and the gut microbiota were monitored. RESULTS: After 8 weeks, the mean body weight of HFD-fed mice was 39.8±1.2 g compared with 35.8±1.3 g for the HFD+1% WEAC group, corresponding to a reduction of 4 g or 10% of body weight (P<0.0001). WEAC supplementation reduced fat accumulation and serum triglycerides in a statistically significant manner in HFD-fed mice. WEAC also reversed the effects of HFD on inflammation markers (interleukin-1ß, interleukin-6, tumor necrosis factor-α), insulin resistance and adipokine production (leptin and adiponectin). Notably, WEAC increased the expression of intestinal tight junctions (zonula occludens-1 and occludin) and antimicrobial proteins (Reg3g and lysozyme C) in the small intestine, leading to reduced blood endotoxemia. Finally, WEAC modulated the composition of the gut microbiota, reducing the Firmicutes/Bacteroidetes ratio and increasing the level of Akkermansia muciniphila and other bacterial species associated with anti-inflammatory properties. CONCLUSIONS: Supplementation with A. cinnamomea produces anti-obesogenic, anti-inflammatory and antidiabetic effects in HFD-fed mice by maintaining intestinal integrity and modulating the gut microbiota.

Prevalence of HIV-1 transmitted drug resistance in the incarcerated population.

OBJECTIVES: The aim of the study was to determine the prevalence of transmitted drug resistance (TDR)-associated mutations among treatment-naïve, incarcerated individuals with HIV-1 infection in the USA as well as the class TDR and antiretroviral (ARV) mutations present at baseline. METHODS: Patients over the age of 18 years were included in the study if they had been diagnosed with HIV infection, if their HIV infection was managed through telemedicine and if they were incarcerated in the State of Illinois Department of Corrections between 10 July 2010 and 29 April 2016. Additionally, the patients were required to have a documented genotype and be ARV-naïve. A medical chart review was conducted to assess demographic information, disease burden, and risk factors for acquiring the virus. RESULTS: The inclusion criteria were met for 105 patients. A total of 24 patients (23%) had a clinically significant mutation associated with resistance to any drug class. The prevalence of mutations conferring clinically significant resistance was 19% for nonnucleoside reverse transcriptase inhibitors (NNRTIs), 18% for nucleoside reverse transcriptase inhibitors (NRTIs), and 4% for protease inhibitors (PIs). Five per cent of patients had dual-class TDR to both NRTI and NNRTI drug classes and 2% of patients had mutations to both NNRTI and PI drug classes. There was no significant increase in the prevalence of clinically relevant drug resistance mutations based on demographics, burden of disease, or risk factors for acquiring the virus. CONCLUSIONS: A high prevalence of TDR was identified in the ARV-naïve incarcerated population. The results of this study indicate an increased prevalence of TDR in a largely unstudied incarcerated population, demonstrating the need for increased monitoring of resistance in HIV-infected patients world-wide.

Towards elucidating the photochemistry of the sunscreen filter ethyl ferulate using time-resolved gas-phase spectroscopy.

Ultrafast time-resolved ion yield (TR-IY) and velocity map imaging spectroscopies are employed to reveal the relaxation dynamics after photoexcitation in ethyl 4-hydroxy-3-methoxycinnamate (ethyl ferulate, EF), an active ingredient in commercially available sunscreens. In keeping with a bottom-up strategy, the building blocks of EF, 2-methoxy-4-vinylphenol (MVP) and 4-hydroxy-3-methoxycinnamyl alcohol (coniferyl alcohol, ConA), were also studied to assist in our understanding of the dynamics of EF as we build up in molecular complexity. In contrast to the excited state dynamics of MVP and ConA, which are described by a single time constant (>900 ps), the dynamics of EF are described by three time constants (15 ± 4 ps, 148 ± 47 ps, and >900 ps). A mechanism is proposed involving internal conversion (IC) between the initially excited S1(11ππ*) and S2(11nπ*) states followed by intramolecular vibrational redistribution (IVR) on both states, in competition with intersystem crossing onto neighbouring triplet states (15 ± 4 ps). IVR and IC within the triplet manifold then ensues (148 ± 47 ps) to populate a low-lying triplet state (>900 ps). Importantly, the fluorescence spectrum of EF at the S1 origin, along with the associated lifetime (6.9 ± 0.1 ns), suggests that population is trapped, during initial IVR, on the S1(11ππ*) state. This serves to demonstrate the complex, competing dynamics in this sunscreen filter molecule.

Torsional Motion of the Chromophore Catechol following the Absorption of Ultraviolet Light.

The ability to probe energy flow in molecules, following the absorption of ultraviolet light, is crucial to unraveling photophysical phenomena. Here we excite a coherent superposition of vibrational states in the first excited electronic state (S1) in catechol, resulting in a vibrational wave packet. The observed quantum beats, assigned to superpositions of the low-frequency, and strongly mixed, O-H torsional mode τ2, elegantly demonstrate how changes in geometry upon photoionization from the S1 state to the ground state of the cation (D0) enables one to probe energy flow at the very early stages of photoexcitation in this biological chromophore.

Probing Rotational Motion in 4-tert-Butylcatechol through H Atom Photofragmentation: Deviations from Axial Recoil.

The time-resolved photofragmentation dynamics of 4-tert-butylcatechol were studied following one photon excitation to the S1 (1(1)ππ*) state with ultraviolet radiation in the range 260 ≤ λ ≤ 286 nm. The preparation of an aligned molecular ensemble via photoexcitation leads to anisotropy in the H atom photofragments. These H atoms originate from the decay of the S1 state through coupling onto the S2 ((1)πσ*) state, which is dissociative along the nonintramolecular hydrogen bonded "free" O-H bond. The degree of anisotropy of these photogenerated H atoms decreases with increasing pump-probe time delay. This is attributed to rotational dephasing of the initially aligned molecular ensemble. The measured dephasing occurs on a time scale akin to the appearance time of these H atoms, which likely places an intrinsic lower bound on the dephasing lifetime. The present work demonstrates how a careful balance between the appearance time of the H atoms, determined by the S1 lifetime, and the rotational dephasing in 4-tert-butylcatechol provides an opportune window to probe rotational motion in real time.

The economic impact of anastomotic leakage after anterior resections in English NHS hospitals: are we adequately remunerating them?

AIM: Our aim was to determine the frequency and economic impact of anastomotic leakage (AL) at local and national levels in England. METHOD: All patients who underwent AR in Oxford between 2007 and 2009 were evaluated for AL. Hospital Episode Statistics (HES) data were used to determine reoperation rates after elective AR (n = 23 388) in England between 2000 and 2008. Hospital episode remuneration costs were calculated by the local commissioning department and compared with Department of Health (DH) reference index costs. RESULTS: The frequency of AL following anterior resection was 10.9% (31 out of 285) in Oxford. Laparotomy for leakage was performed in 5.6% of cases. The 30-day hospital mortality rate for all ARs was 2.1%, compared with 3.2% after AL. The national relaparotomy rate (within 28 days) and 30-day hospital mortality in English National Health Service (NHS) trusts following AR were 5.9% and 2.9%, respectively. Institutional remunerated tariffs (£6233 (SD ± 965)) were similar to DH reference costs (£6319 (SD ± 1830)) after uncomplicated AR. However, there was a significant (P = 0.008) discrepancy between the remunerated tariff for AL (£9605 (SD ± 6908)) and the actual cost (£17 220 (SD ± 9642)). AL resulted in an additional annual cost of approximately £1.1 million to £3.5 million when extrapolated nationally. CONCLUSION: The estimated economic burden of anastomotic leakage following AR is approximately double that of the remunerated tariff.

Contactless skin perfusion monitoring with video cameras: tracking pharmacological vasoconstriction and vasodilation using photoplethysmographic changes.

Objectives.Clinical assessment of skin perfusion informs prognosis in critically ill patients. Video camera monitoring could provide an objective, continuous method to monitor skin perfusion. In this prospective, interventional study of healthy volunteers, we tested whether video camera-derived photoplethysmography imaging and colour measurements could detect drug-induced skin perfusion changes.Approach.We monitored the lower limbs of 30 volunteers using video cameras while administering phenylephrine (a vasoconstrictor) and glyceryl trinitrate (a vasodilator). We report relative pixel intensity changes from baseline, as absolute values are sensitive to environmental factors. The primary outcome was the pre- to peak- infusion green channel amplitude change in the pulsatile PPGi waveform component. Secondary outcomes were pre-to-peak changes in the photoplethysmographic imaging waveform baseline, skin colour hue and skin colour saturation.Main results.The 30 participants had a median age of 29 years (IQR 25-34), sixteen (53%) were male. A 34.7% (p= 0.0001) mean decrease in the amplitude of the pulsatile photoplethysmographic imaging waveform occurred following phenylephrine infusion. A 30.7% (p= 0.000004) mean increase occurred following glyceryl trinitrate infusion. The photoplethysmographic imaging baseline decreased with phenylephrine by 2.1% (p= 0.000 02) and increased with glyceryl trinitrate by 0.5% (p= 0.026). Skin colour hue changed in opposite direction with phenylephrine (-0.0013,p= 0.0002) and glyceryl trinitrate (+0.0006,p= 0.019). Skin colour saturation decreased with phenylephrine by 0.0022 (p= 0.0002), with no significant change observed with glyceryl trinitrate (+0.0005,p= 0.21).Significance.Drug-induced vasoconstriction and vasodilation are associated with detectable changes in photoplethysmographic imaging waveform parameters and skin hue. Our findings suggest video cameras have great potential for continuous, contactless skin perfusion monitoring.

Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs.

In most mammalian cells nucleoside uptake occurs primarily via broad-specificity, es (e, equilibrative; 5, sensitive to NBMPR inhibition) transporters that are potently inhibited by nitrobenzylthioinosine (NBMPR). These transporters are essential for nucleotide synthesis by salvage pathways in hemopoietic and other cells that lack de novo pathways and are the route of cellular uptake for many cytotoxic nucleosides used in cancer and viral chemotherapy. They play an important role in adenosine-mediated regulation of many physiological processes, including neurotransmission and platelet aggregation, and are a target for coronary vasodilator drugs. We have previously reported the purification of the prototypic es transporter from human erythrocytes and have shown that this glycoprotein of apparent M, 55,000 is immunologically related to nucleoside transporters from several other species and tissues, including human placenta. Here we report the isolation of a human placental cDNA encoding a 456-residue glycoprotein with functional characteristics typical of an es-type transporter. It is predicted to possess 11 membrane-spanning regions and is homologous to several proteins of unknown function in yeast, nematodes, plants and mammals. Because of its central role in the uptake both of adenosine and of chemotherapeutic nucleosides, study of this protein should not only provide insights into the physiological roles of nucleoside transport but also open the way to improved therapies.

Thymosin beta 4 sulfoxide is an anti-inflammatory agent generated by monocytes in the presence of glucocorticoids.

The possibility that glucocorticoids upregulate the expression of anti-inflammatory mediators is an exciting prospect for therapy in inflammatory diseases, because these molecules could give the therapeutic benefits of steroids without toxic side effects. Supernatants from monocytes and macrophages cultured in the presence of glucocorticoids increase the dispersion of neutrophils from a cell pellet in the capillary tube migration assay. This supernatant factor, unlike other neutrophil agonists, promotes dispersive locomotion of neutrophils at uniform concentration, lowers their adhesion to endothelial cells, inhibits their chemotactic response to fMLP and induces distinctive morphological changes. Here we show that thymosin beta4 sulfoxide is generated by monocytes in the presence of glucocorticoids and acts as a signal to inhibit an inflammatory response. In vitro, thymosin beta4 sulfoxide inhibited neutrophil chemotaxis, and in vivo, the oxidized peptide, but not the native form, was a potent inhibitor of carrageenin-induced edema in the mouse paw. Thymosin beta4 is unique, because oxidation attenuates its intracellular G-actin sequestering activity, but greatly enhances its extracellular signaling properties. This description of methionine oxidation conferring extracellular function on a cytosolic protein may have far-reaching implications for future strategies of anti-inflammatory therapy.

A multi-institutional randomized controlled trial comparing first-generation transrectal high-resolution micro-ultrasound with conventional frequency transrectal ultrasound for prostate biopsy.

Objectives: To study high-frequency 29 MHz transrectal side-fire micro-ultrasound (micro-US) for the detection of clinically significant prostate cancer (csPCa) on prostate biopsy, and validate an image interpretation protocol for micro-US imaging of the prostate. Materials and methods: A prospective randomized clinical trial was performed where 1676 men with indications for prostate biopsy and without known prostate cancer were randomized 1:1 to micro-US vs conventional end-fire ultrasound (conv-US) transrectal-guided prostate biopsy across five sites in North America. The trial was split into two phases, before and after training on a micro-US image interpretation protocol that was developed during the trial using data from the pre-training micro-US arm. Investigators received a standardized training program mid-trial, and the post-training micro-US data were used to examine the training effect. Results: Detection of csPCa (the primary outcome) was no better with the first-generation micro-US system than with conv-US in the overall population (34.6% vs 36.6%, respectively, P = .21). Data from the first portion of the trial were, however, used to develop an image interpretation protocol termed PRI-MUS in order to address the lack of understanding of the appearance of cancer under micro-US. Micro-US sensitivity in the post-training group improved to 60.8% from 24.6% (P < .01), while specificity decreased (from 84.2% to 63.2%). Detection of csPCa in the micro-US arm increased by 7% after training (32% to 39%, P < .03), but training instituted mid-trial did not affect the overall results of the comparison between arms. Conclusion: Micro-US provided no clear benefit over conv-US for the detection of csPCa at biopsy. However, it became evident during the trial that training and increasing experience with this novel technology improved the performance of this first-generation system.

Identification and characterization of a pore-forming protein of human peripheral blood natural killer cells.

We show here that human peripheral blood NK cells contain a pore-forming protein (PFP) with an Mr of 70,000-72,000 that assembles structural lesions (with an average internal diameter of 150-170 A) and forms functional channels. The PFP was isolated by affinity chromatography from human NK cells, using a specific anti-C9 antiserum as the immunoadsorbent. The NK cells were isolated from PBL by positive or negative selection by indirect rosetting using a panel of monoclonal antibodies directed against different NK and T cell surface antigens. PFP was identified in NK cells freshly isolated and isolated from cultured PBL, both stimulated with interleukin 2, but not in NK cell-depleted lymphocytes. In planar bilayers, the channels formed by the NK cell-derived PFP are highly voltage resistant, with most channels persisting in the open state once they have inserted into the bilayer. The unit conductances of these channels range 0.3-1 nS in 0.1 M NaCl. The channels show poor selectivity for monovalent and divalent ions. The PFP is also released from human NK cells stimulated with the calcium ionophore A23187, suggesting that this protein, like the one produced by murine CTL lines, may be similarly secreted during cell-mediated killing. Its identification in primary human NK cell cultures indicates that this protein may play an active role in NK cell-mediated killing.

The involvement of interleukin (IL)-15 in regulating the differentiation of granulated metrial gland cells in mouse pregnant uterus.

Previous studies have suggested that granulated metrial gland (GMG) cells are bone marrow-derived lymphoid cells, which differentiate in situ in the mouse pregnant uterus into natural killer (NK)-like cells. Similar to NK cells, GMG cells express an abundant level of cytolytic mediators such as perforin. The factor(s) regulating the differentiation of GMG cells remain(s) to be identified, although cytokines previously implicated in the stimulation/activation of NK cells (e.g., IL-2, IL-6, IL-7, and IL-12) can be considered as potential candidates. Recently, IL-15, a novel cytokine, which displays biological activities similar to IL-2, has also been shown to be capable of activating NK cells. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, we have demonstrated in the present study that IL-15 and its cognate receptor, but not the other cytokines, are expressed in the mouse pregnant uterus, with a time course concomitant with those of cytolytic mediators in differentiated GMG cells. Moreover, IL-15, though not IL-2, is capable of inducing the expression of perforin and granzymes in pregnant uterine tissues explanted in vitro. Data obtained from in situ hybridization study have suggested that the macrophages present in the pregnant uterus may be responsible for the production of IL-15. These results suggest that IL-15 is involved in regulating the differentiation of GMG cells during mouse pregnancy.

Properties of a purified pore-forming protein (perforin 1) isolated from H-2-restricted cytotoxic T cell granules.

Histocompatibility-restricted cytotoxic T lymphocytes produce circular lesions on target cell membranes. The pore-forming protein (PFP or perforin 1) that forms these membrane lesions has been purified from lymphocytes. At 37 degrees C, in the presence of Ca2+, this protein polymerizes into a supramolecular tubular complex of Mr greater than 10(6) that partially resists dissociation by SDS and reducing agents. It incorporates spontaneously into planar lipid bilayers during polymerization to form nonselective ion channels, showing heterogeneous size distribution, the smallest conductance per unit being identified as 400 pS in 0.1 M NaCl. PFP/P1 that had been assembled in lipid vesicles before incorporation into planar bilayer show much larger single channel conductance, ranging from 1 to 6 nS in 0.1 M NaCl, suggesting that PFP/P1 may assume multiple functional sizes in proportion to its state of polymerization. The reconstituted channels are relatively voltage-insensitive, with most channels persisting in the open state for seconds to minutes. Nucleated cells are rapidly depolarized by this protein. The purified protein lyses a variety of tumor cells. Polymerization and functional channel activity are absolutely Ca2+-dependent. The activity of this protein may play a direct role in T lymphocyte-mediated cytolysis.

Perforin and serine esterase gene expression in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A.

A pore-forming protein (PFP; perforin) and various serine esterases (SE) have been identified in the cytoplasmic granules of CTL and NK cells. Perforin and several SE have recently been cloned. Northern blotting analysis was performed here using cDNA probes specific for human perforin and two SE (SE 1/HS and SE 2/GB) to monitor the levels of specific mRNAs in mitogen-stimulated primary human T cells. These mRNAs were rapidly induced by IL-2 with optimal responses at 300 U/ml. After IL-2 treatment, mRNAs for perforin, SE 1, and SE 2 peaked at 12-24 h and decreased after 48 h. The three mRNAs were also induced in T cells treated with a combination of PMA plus lectin, OKT3 mAb, or plastic-adherent accessory cells. However, the induction induced by PMA/mitogen followed a slower kinetics, peaking at 48 h. In general, we found that SE 1 mRNA was more readily induced by IL-2, while SE 2 responded better to PMA/mitogen. Similar patterns of mRNA expression were observed for both unprimed T cells and PHA-primed T blasts. After stimulation with IL-2 and PMA/mitogen, the T8+ subset was shown to be the main producer of perforin, SE 1, and SE 2. Low levels of all three mRNAs, however, were also detected in the T4+ subset. The induction of all three mRNAs by either IL-2 or PMA/mitogen was partially blocked by the immunosuppressive drug cyclosporin A (CsA), but not by the biologically inactive analogue cyclosporin H. Together, these results point to some similarities and differences with upregulation of granule mediator mRNAs relative to lymphokine mRNAs. Both sets of genes require two signals for their induction by mitogens. In contrast to lymphokines, there is a strong response of granule mRNAs to IL-2, and the induction of these transcripts is only partially blocked by CsA.

Characterization of a membrane pore-forming protein from Entamoeba histolytica.

We describe the partial purification and characterization of a pore-forming material (PEM) from Entamoeba histolytica. The formation of ion channels by PFM was examined in three systems. (a) PFM depolarizes J774 macrophages and mouse spleen lymphocytes as measured by [3H]TPP+ uptake. (b) PFM induces rapid monovalent cation flux across the membrane of phosphatidylcholine-cholesterol vesicles. (c) PFM confers a voltage-dependent conductance to artificial planar bilayers, which is resolved as a summation of opening of individually conducting steps of 67 pS in 0.1 M KCl. Monomers of PFM are functional; however, a preferential aggregation occurs in the planar bilayer. Activity is pronase, trypsin, and heat sensitive and is stable between pH 5-8. PFM is not secreted by unstimulated amoebae but after exposure to the calcium ionophore A23187, concanavalin A, and E. coli lipopolysaccharide, 5-10% of the total cell content of PFM is released into the medium within 5-10 min. High-performance gel filtration results in an approximately 1,000-fold purification of PFM and gives an Mr of 30,000. This protein may play a role in the cytotoxicity mediated by E. histolytica.

A calcium- and perforin-independent pathway of killing mediated by murine cytolytic lymphocytes.

Cytotoxic T lymphocytes have been thought to lyse cellular targets in the past by a calcium-dependent pathway. This notion was recently supported by the identification and purification of a pore-forming protein (perforin) from the granules of these cell types. Here, we show that perforin is absent from a number of cell lines that nevertheless display vigorous cytolytic activity toward target cells. The cytotoxic activity of eight murine CTL lines is completely or partially retained in the absence of calcium. The calcium-independent lytic activity is associated with two subcellular fraction peaks isolated by Percoll gradient centrifugation, e.g., a heavy density band migrating with granule markers and a lighter band corresponding to free cytosolic material. These results suggest a complex picture of lymphocyte-mediated killing involving probably multiple mechanisms and mediators that may operate in concert or independently in the delivery of the lethal hit.