Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot.

Background: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis on whether the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. Methods: Mice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. Results: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted. Conclusion: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

Characterisation, identification, clustering, and classification of disease.

The importance of quantifying the distribution and determinants of multimorbidity has prompted novel data-driven classifications of disease. Applications have included improved statistical power and refined prognoses for a range of respiratory, infectious, autoimmune, and neurological diseases, with studies using molecular information, age of disease incidence, and sequences of disease onset ("disease trajectories") to classify disease clusters. Here we consider whether easily measured risk factors such as height and BMI can effectively characterise diseases in UK Biobank data, combining established statistical methods in new but rigorous ways to provide clinically relevant comparisons and clusters of disease. Over 400 common diseases were selected for analysis using clinical and epidemiological criteria, and conventional proportional hazards models were used to estimate associations with 12 established risk factors. Several diseases had strongly sex-dependent associations of disease risk with BMI. Importantly, a large proportion of diseases affecting both sexes could be identified by their risk factors, and equivalent diseases tended to cluster adjacently. These included 10 diseases presently classified as "Symptoms, signs, and abnormal clinical and laboratory findings, not elsewhere classified". Many clusters are associated with a shared, known pathogenesis, others suggest likely but presently unconfirmed causes. The specificity of associations and shared pathogenesis of many clustered diseases provide a new perspective on the interactions between biological pathways, risk factors, and patterns of disease such as multimorbidity.

Delivery of gelfoam-enabled cells and vectors into the pericardial space using a percutaneous approach in a porcine model.

Intrapericardial drug delivery is a promising procedure, with the ability to localize therapeutics with the heart. Gelfoam particles are nontoxic, inexpensive, nonimmunogenic and biodegradable compounds that can be used to deliver therapeutic agents. We developed a new percutaneous approach method for intrapericardial injection, puncturing the pericardial sac safely under fluoroscopy and intravascular ultrasound (IVUS) guidance. In a porcine model of myocardial infarction (MI), we deployed gelfoam particles carrying either (a) autologous mesenchymal stem cells (MSCs) or (b) an adenovirus encoding enhanced green fluorescent protein (eGFP) 48 h post-MI. The presence of MSCs and viral infection at the infarct zone was confirmed by immunoflourescence and PCR. Puncture was performed successfully in 16 animals. Using IVUS, we successfully determined the size of the pericardial space before the puncture, and safely accessed that space in setting of pericardial effusion and also adhesions induced by the MI. Intrapericardial injection of gelfoam was safe and reliable. Presence of the MSCs and eGFP expression from adenovirus in the myocardium were confirmed after delivery. Our novel percutaneous approach to deliver (stem-) cells or adenovirus was safe and efficient in this pre-clinical model. IVUS-guided delivery is a minimally invasive procedure that seems to be a promising new strategy to deliver therapeutic agents locally to the heart.

Brain: non-infective and non-neoplastic manifestations of HIV.

The aim of this pictorial review is to display a wide range of non-infective non-neoplastic pathologies such as demyelinating disorders (including the immune reconstitution inflammatory syndrome, tumefactive demyelination and HIV encephalopathy), Parkinson's disease, vascular disorders (including strokes, vasculitis and dural venous sinus thrombosis) and spontaneous subdural bleeding.

Deglutitive laryngeal closure in stroke patients.

BACKGROUND: Dysphagia has been reported in up to 70% of patients with stroke, predisposing them to aspiration and pneumonia. Despite this, the mechanism for aspiration remains unclear. AIMS: To determine the relationship between bolus flow and laryngeal closure during swallowing in patients with stroke and to examine the sensorimotor mechanisms leading to aspiration. METHODS: Measures of swallowing and bolus flow were taken from digital videofluoroscopic images in 90 patients with stroke and 50 healthy adults, after repeated volitional swallows of controlled volumes of thin liquid. Aspiration was assessed using a validated Penetration-Aspiration Scale. Oral sensation was also measured by electrical stimulation at the faucial pillars. RESULTS: After stroke, laryngeal ascent was delayed (mean (standard deviation (SD)) 0.31 (0.06) s, p<0.001), resulting in prolongation of pharyngeal transit time (1.17 (0.07) s, p<0.001) without a concomitant increase in laryngeal closure duration (0.84 (0.04) s, p = 0.9). The delay in laryngeal elevation correlated with both the severity of aspiration (r = 0.5, p<0.001) and oral sensation (r = 0.5, p<0.001). CONCLUSIONS: After stroke, duration of laryngeal delay and degree of sensory deficit are associated with the severity of aspiration. These findings indicate a role for sensorimotor interactions in control of swallowing and have implications for the assessment and management of dysphagia after stroke.

The imaging appearances of intracranial CNS infections in adult HIV and AIDS patients.

The spectrum of pathology affecting the central nervous system (CNS) in patients suffering from acquired immunodeficiency syndrome (AIDS) is broad and comprises predominantly opportunistic infections and neoplasms. It is estimated that approximately one-third of all patients with AIDS develop neurological complications. The organisms responsible for AIDS are human retroviruses: primarily the human immunodeficiency virus type 1 (HIV). In this review we shall focus on the neurological complications of HIV and AIDS which are applicable to the more frequently occurring intracranial infective organisms. Attention will be paid specifically to those CNS manifestations occurring in the adult HIV and AIDS population as infection in the paediatric HIV and AIDS group, although bearing some similarities, demonstrates some important differences.

Reorientation of aquaporin-1 topology during maturation in the endoplasmic reticulum.

The topology of most eukaryotic polytopic membrane proteins is established cotranslationally in the endoplasmic reticulum (ER) through a series of coordinated translocation and membrane integration events. For the human aquaporin water channel AQP1, however, the initial four-segment-spanning topology at the ER membrane differs from the mature six-segment-spanning topology at the plasma membrane. Here we use epitope-tagged AQP1 constructs to follow the transmembrane (TM) orientation of key internal peptide loops in Xenopus oocyte and cell-free systems. This analysis revealed that AQP1 maturation in the ER involves a novel topological reorientation of three internal TM segments and two peptide loops. After the synthesis of TMs 4-6, TM3 underwent a 180-degree rotation in which TM3 C-terminal flanking residues were translocated from their initial cytosolic location into the ER lumen and N-terminal flanking residues underwent retrograde translocation from the ER lumen to the cytosol. These events convert TM3 from a type I to a type II topology and reposition TM2 and TM4 into transmembrane conformations consistent with the predicted six-segment-spanning AQP1 topology. AQP1 topological reorientation was also associated with maturation from a protease-sensitive conformation to a protease-resistant structure with water channel function. These studies demonstrate that initial protein topology established via cotranslational translocation events in the ER is dynamic and may be modified by subsequent steps of folding and/or maturation.

Identification of sequence determinants that direct different intracellular folding pathways for aquaporin-1 and aquaporin-4.

Homologous aquaporin water channels utilize different folding pathways to acquire their transmembrane (TM) topology in the endoplasmic reticulum (ER). AQP4 acquires each of its six TM segments via cotranslational translocation events, whereas AQP1 is initially synthesized with four TM segments and subsequently converted into a six membrane-spanning topology. To identify sequence determinants responsible for these pathways, peptide segments from AQP1 and AQP4 were systematically exchanged. Chimeric proteins were then truncated, fused to a C-terminal translocation reporter, and topology was analyzed by protease accessibility. In each chimeric context, TM1 initiated ER targeting and translocation. However, AQP4-TM2 cotranslationally terminated translocation, while AQP1-TM2 failed to terminate translocation and passed into the ER lumen. This difference in stop transfer activity was due to two residues that altered both the length and hydrophobicity of TM2 (Asn(49) and Lys(51) in AQP1 versus Met(48) and Leu(50) in AQP4). A second peptide region was identified within the TM3-4 peptide loop that enabled AQP4-TM3 but not AQP1-TM3 to reinitiate translocation and cotranslationally span the membrane. Based on these findings, it was possible to convert AQP1 into a cotranslational biogenesis mode similar to that of AQP4 by substituting just two peptide regions at the N terminus of TM2 and the C terminus of TM3. Interestingly, each of these substitutions disrupted water channel activity. These data thus establish the structural basis for different AQP folding pathways and provide evidence that variations in cotranslational folding enable polytopic proteins to acquire and/or maintain primary sequence determinants necessary for function.

Long term outcome of unilateral pallidotomy: follow up of 15 patients for 3 years.

OBJECTIVES: With the advent of new antiparkinsonian drug therapy and promising results from subthalamic and pallidal stimulation, this study evaluated the long term efficacy of unilateral pallidotomy, a technique which has gained popularity over the past decade for the management of advanced Parkinson's disease. METHODS: The 15 patients reported here are part of the original cohort of 24 patients who underwent posteroventral pallidotomy for motor fluctuations and disabling dyskinesias 3 years ago as part of a prospective study. Evaluation scales included the unified Parkinson's disease rating scale, the Goetz dyskinesia scale, and the Purdue pegboard test. RESULTS: When compared with the prepallidotomy scores, the reduction in the limb dyskinesias and off state tremor scores persisted on the side contralateral to pallidotomy at the end of 3 years (dyskinesias were reduced by 64% (p<0.01) and tremor by 63% (p<0.05). Other measures tended to deteriorate. The dosage of antiparkinsonian medications did not change significantly from 3 months prepallidotomy to 3 years postpallidotomy. CONCLUSIONS: Although unilateral pallidotomy is useful in controlling the contralateral dyskinesias and tremor 3 years after surgery, all other early benefits disappear and activities of daily living continue to worsen.

Reassessment of unilateral pallidotomy in Parkinson's disease. A 2-year follow-up study.

Unilateral pallidotomy has gained popularity in treating the motor symptoms of Parkinson's disease. We present the results of a 2-year post-pallidotomy follow-up study. Using the Unified Parkinson's Disease Rating Scale (UPDRS), the Goetz dyskinesia scale and the Purdue Pegboard Test (PPBT), we evaluated 20 patients at regular intervals both off and on medications for 2 years post-pallidotomy. There were no significant changes in the dosages of antiparkinsonian medications from 3 months pre-pallidotomy to 2 years post-pallidotomy. On the side contralateral to the operation, the improvements were preserved in 'on'-state dyskinesia (83% reduction from pre-pallidotomy to 2 years post-pallidotomy, P < 0.001) and 'off'-state tremor (90% reduction from pre-pallidotomy to 2 years post-pallidotomy, P = 0.005). There were no statistically significant differences between pre-pallidotomy scores and those at 2 years post-pallidotomy in ipsilateral dyskinesia, axial dyskinesia, 'off'- or 'on'-state PPBT, 'off'-state Activities of Daily Living (ADL) and 'off'-state gait and postural stability. After 2 years, the 'on'-state ADL scores worsened by 75%, compared with pre-pallidotomy (P = 0.005). We conclude that 2 years after pallidotomy, the improvements in dyskinesia and tremor on the side contralateral to pallidotomy are preserved, while the initial improvements in most other deficits disappear, either because of progression of pathology or loss of the early efficacy achieved by surgery.

Efficacy, stability and predictors of outcome of pallidotomy for Parkinson's disease. Six-month follow-up with additional 1-year observations.

We tested the efficacy, stability and predictors of outcome of unilateral pallidotomy used to treat patients with Parkinson's disease inadequately controlled with pharmacotherapy (IP). The surgical procedure was as simple as possible; we used CT rather than MRI, and we omitted microelectrode recording. We studied 24 patients with IP; 22 of these patients had drug-induced dyskinesias. There was a significant and stable improvement in all the major parkinsonian motor signs in the OFF (medication) state on the contralateral side. In the ON (medication) state peak-dose dyskinesias were alleviated on the contralateral side. The only significant and stable change on the ipsilateral side was improvement in dyskinesias less marked than on the contralateral side. The improvement in Unified Parkinson's Disease Rating Scale motor scores in the OFF state increased with age. The improvement in total dyskinesia scores occurred irrespective of age, but increased with duration of disease, duration of dyskinesias and baseline severity of dyskinesias. Five patients had transient neurological complications while facial paresis was permanent in one subject. Our results are similar to those obtained by others who used the time consuming microelectrode recording technique for localization. By simplifying the procedure in the way that we describe, the operation could become available to a greater number of patients.

Occult spinal dysraphism--the common combination of lesions and the clinical manifestations in 50 patients.

Fifty patients (26 males, 24 females) aged from birth to 51 years with occult spinal dysraphism were identified. All had Magnetic Resonance (MR) scans carried out. The MR scans were examined to determine the vertebral level of the conus and to see if one or more of the following were present; lipoma, syrinx, dermoid, diastematomyelia, and meningocele. In 43 patients the conus lay below the level of L3, 23 had a lipoma, 23 a meningocele, 20 an open central canal in the spinal cord or a syrinx, 15 a diastematomyelia and 4 a dermoid. The commonest combination of lesions was a long cord, a syrinx or an open central canal in the spinal cord and a lipoma. The diastematomyelias were always associated with a long cord and had the highest incidence of vertebral body anomalies (60%). The most frequent recorded signs were deformities of the feet, short legs, wasting of the calf muscles, weakness of the legs, back pain and bladder dysfunction. Combinations of these occurred with all of the lesions although some had none. Whilst deterioration was seen in about half of the patients, acute deterioration was uncommon and was associated most frequently with a dermoid or an expanded syrinx.

Investigation of the uptake of drugs by individual cells using a scanning proton microprobe (SPM).

In this paper we demonstrate the use of micro-PIXE (proton induced X-ray emission) for measuring the quantitative uptake of anti-AIDS drugs, containing metal atoms, by individual Vero cells (African green monkey kidney cell line). Hetero-polytungstates, which are assessed to present an activity against the HIV virus, were studied using Vero cells. It was found that unlike other techniques, SPM offers both the sensitivity and the spatial resolution to carry out these programs of investigations. The use of elemental analysis in single cells of cultured cell lines has shown to have distinct advantages over peripheral blood lymphocytes.

[Pediatric nosocomial bacteremia. Potential usefulness of culturing infusion liquids]. / Bacteriemia nosocomial pediátrica. Utilidad potencial del cultivo de los líquidos de infusión.

Hospital-acquired bacteremia is a common cause of morbidity and mortality, mainly in pediatric units. In a 25 month retrospective study, we analyzed the blood cultures from the Hospital General Regional of the city of Leon, Guanajuato State, Mexico, in order to establish the causal agents of nosocomial bacteremia and infer some associations with contaminated intravenous infusion fluids. In addition we performed a two month study to culture the flasks and intravenous tubing used in our infusions. Five hundred and fifty one blood cultures drawn from August 1990 to September 1992 were analyzed. A total of 135 (24.5%) were positive, most of them (51.8%) with strains of the Klebsielleae tribe (SKT) (Klebsiella, Enterobacter, Serratia). The global incidence of bacteremia in the two year period was 4.3%. In the infusion study, 230 intravenous fluids were cultured, with 68 isolates (30%) most from the SKT tribe. A final consideration is made on the role that inadequate management of intravenous liquids could play in the development of endemic and epidemic nosocomial bacteremia in our hospital, and the eventual utility of making cultures of the i.v. liquids.

Timing of surgery in relation to the menstrual cycle in premenopausal women with operable breast cancer.

Recent studies have suggested that the timing of surgery in relation to the menstrual cycle might influence survival of premenopausal women with operable breast cancer. The data of 96 premenopausal patients who underwent primary surgery for operable breast carcinoma between 1975 and 1988 were analysed. At 10 years, disease-free and overall survival rates of patients whose initial surgery was 1-12 days after the starting date of the last menstrual period (follicular phase) were significantly poorer compared with survival of those who underwent operation more than 12 days after the last menstruation (luteal phase) (disease-free survival rate 40 versus 72 per cent, P = 0.002; overall survival rate 40 versus 79 per cent, P = 0.001). These differences in survival remained significant in a second analysis based on the menstrual phase at the time of both initial and definitive operation. Menstrual phase had the greatest impact on the survival of patients with positive axillary nodes (P = 0.009). Prospective studies are required to elucidate the relationship between the timing of all surgical procedures during the menstrual cycle and survival.

Timing of surgery influences survival in receptor-negative as well as receptor-positive breast cancer.

Analysis of oestrogen and progesterone receptor (ER, PR) status was interpreted in relation to menstrual phase at the time of surgery and survival in 84 women diagnosed with breast cancer between 1975 and 1988. We showed previously (Br J Surgery 1994, 81, 217-220) that long-term survival was significantly poorer when surgery was performed during the follicular phase of the menstrual cycle compared to luteal phase; we now demonstrate that this effect on survival is at least as important in receptor-negative as receptor-positive patients. At 10 years, overall survival (OS) of ER-positive patients who had their biopsy during the follicular phase was significantly poorer than for those whose biopsy was performed during the luteal phase of their menstrual cycle (52 versus 88%, P = 0.02). OS for the ER-negative follicular phase group was also significantly poorer than that for the ER-negative luteal phase group (33 versus 76%, P = 0.009). The OS difference between the PR-positive follicular phase group and PR-positive luteal phase group was of borderline significance (60 versus 87%, P = 0.06), while the difference in OS between the PR-negative follicular phase group and that of the PR-negative luteal phase group was highly significant (13 versus 76%, P = 0.001). Disease-free survival for these groups followed a similar trend. The survival differences in receptor-negative women suggest that hormonal fluctuations at the time of surgery may have complex indirect effects on tumour growth and metastasis. The mechanism, if indeed independent of the tumour steroid receptors, could also apply in other cancers.