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.

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.

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.

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.

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.

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.

Systemic immunization of sheep with surface antigens from Haemonchus contortus larvae.

Sheep immunized systemically with a surface extract from third-stage H. contortus larvae showed high serum antibody reactivity against surface antigens and whole, viable larvae. After a first infection, no significant difference was found between the mean egg counts of the vaccinates and controls although most vaccinated sheep seemed to show an increased susceptibility to infection. The local abomasal response was stimulated by giving both vaccinated and control sheep a large, abbreviated infection cured after 11 days by drenching. Thereafter, a second challenge infection was given. This immunization regime resulted in seven of the nine vaccinated sheep showing clear protection against the second challenge infection.

Isotope cerebral perfusion studies before and after extracranial/intracranial bypass.

A quick, inexpensive, and reliable method of estimating cerebral perfusion is described that uses simple gamma camera and computer facilities readily available in almost all neuroradiology departments. Eighty-eight patients with unilateral cerebral ischemia were studied who satisfied the criteria for inclusion in the international cooperative study for extracranial/intracranial arterial anastomosis. The results of their isotope cerebral perfusion studies were compared with their computed tomographic scans and cerebral angiograms. The aims of the study were: (1) to ascertain whether the isotope study could predict the presence of a stenosis or occlusion; (2) to determine whether it could forecast the presence of a collateral blood flow and allow an assessment of the contribution of the collaterals to the overall cerebral perfusion; and (3) to assess the patency of the extracranial/intracranial bypass and monitor the effects of that procedure on hemisphere perfusion. The results show that this examination does predict the angiographic findings in nearly all patients and provides useful information about the natural collateral blood flow so that appropriate surgical treatment can be planned for the patient. The changes in cerebral perfusion after bypass were correlated with postoperative angiograms and offer a possible means of assessing and monitoring the effects of revascularization surgery and its contribution to the perfusion of that hemisphere.

Transplantation of human sympathetic neurons and adrenal chromaffin cells into parkinsonian monkeys: no reversal of clinical symptoms.

Cultured human fetal sympathetic ganglion explants or adrenal chromaffin cell aggregates were implanted into the left striatum of monkeys whose left nigrostriatal pathway had been lesioned with the neurotoxin MPTP. There was no clinical reversal of parkinsonian symptoms and PET scans did not show increased striatal fluorodopa uptake from pre-implant levels. At sacrifice, left striatal contents of dopamine were not statistically different from MPTP-treated but non-implanted controls. Histological examinations revealed pockets of extrinsic cells which were found at the end of needle tracks. There was no evidence of immune rejection. The extrinsic cells did not stain for tyrosine hydroxylase or neurofilament, suggesting that they were not dopaminergic neurons. The failure to reverse clinical parkinsonian symptoms highlights the stage of infancy of neural implantation in Parkinson's disease.

Microangiographic study of experimental spinal cord injuries.

The pathology of spinal cord injury has been studied in 34 rabbits and 5 dogs with attention focused on the condition of the microvasculature during the evolution of neuronal and axonal degeneration and necrosis. The animals were killed and perfused arterially with colloidal barium from 10 min to 14 days after a controlled spinal injury. Microradiographs of the injured tissues were obtained and compared with corresponding histological sections. Microangiography at 7 to 14 days defines two zones in the injured spinal cord. Zone 1 is located in the posterocentral part of the cord. Capillaries in this region progressively lose their ability to conduct blood and perfusate over the first 4 hours. Degenerative changes in neurons are visible by 1 hour after injury. Necrosis of all elements including capillaries ensues. Zone 2 surrounds Zone 1. Microvascular patterns are normal in Zone 2 although neuronal and axonal degeneration is severe. Pericapillary hemorrhages which occur as early as 10 min after injury in Zone 1 and become progressively larger over the first 4 hours seldom are seen in Zone 2. The evidence indicates that at all times in the pathogenesis of spinal cord injury the microvasculature in Zone 2 is capable of perfusion. Degeneration of neural structures either precedes microvascular breakdown (Zone 1) or occurs in the absence of microvascular disruption (Zone 2). Recovery of damaged neurons and axons depends upon a preserved microcirculation.

Thalamic stimulation for neuropathic pain.

Eighteen patients with neuropathic pain underwent thalamic electrode implantation. Satisfactory initial pain relief ensued in 14, and their electrode systems were internalized for long-term use. Twelve of the 14 continue to obtain either complete or partial pain relief by regular stimulation. One of the other two patients has had a complete remission of pain, apparently spontaneously, and the other had to have the electrodes removed after it retracted from his thalamus on two occasions. The electrodes have been placed in the sensory nucleus of the thalamus where stimulation evokes paresthesias in the painful part of the body. Technical problems consisting of our inability to locate the target in two patients and our failure to fix the electrode adequately in one prevented us from employing the treatment in three patients. The fourth patient had temperature dysesthesia which was not altered during 2 weeks of stimulation.

CT observations on the natural history of asymptomatic cerebral infarction following transient ischaemic attacks.

Two hundred and sixty-one neurologically asymptomatic patients were examined by computed tomography (CT) following a variable number of transient ischaemic attacks (TIAs) experienced over different time intervals. Cerebral infarcts were discovered in 101 patients. Dominant hemisphere infarcts were usually small, of recent origin and most often associated with less than 5 TIAs and a short history; whereas non-dominant hemisphere infarcts were found twice as frequently and were larger and older and correlated with repeated TIAs over many months. All infarcts, independent of size, were confined to a single vascular territory, suggesting haemodynamic rather than embolic causes. By comparing the age and size of the infarcts with the number of TIAs and their chronicity, the hypothesis emerged, suggesting that infarcts may occur early on and progressively enlarge with repeated attacks.

Comparison of healing of lesions in the calvarium of foetal lambs and young sheep.

This study compares the healing of lesions made in the occipital region of the calvarium of 5 foetal and 4 post-natal lambs. The foetuses, operated on between the 83rd and 91st day of gestation, had a bone flap elevated and the post-natal lambs, aged about 6 weeks, had a craniectomy and replacement of the bone pieces. The foetuses were delivered spontaneously at full-term. All the animals had computed tomographic (CT) scans and, after sacrifice, naked eye examination of the bony lesions. These showed that there was poor ossification of the lesions made in the foetuses compared to those made in the post-natal lambs. These findings need to be taken into account when intra-uterine correction of cranio-facial deformities are being contemplated.

Computed tomographic pointers to the prognosis of subarachnoid haemorrhage.

Ninety-three patients with subarachnoid haemorrhage were studied by computed tomography to seek features which might influence prognosis. In this series a normal scan pointed to complete recovery. The presence of a focal area of low attenuation, whether alone or in association with an haematoma, affected the outcome adversely. Prognosis was also influenced by the size of the haematoma. None of the other findings appeared to be a guide to prognosis.

Bilateral extradural hematoma extending from the foramen magnum to the vertex.

We present a 35-year-old man with an extradural hematoma extending bilaterally across the midline from the foramen magnum to the vertex who completely recovered following surgery. Because of the extent of the fluid collection, computed tomographic scanning proved invaluable in facilitating the planning of our surgical approach. He had a long history of alcohol abuse and there were features suggesting preexisting alcoholic brain atrophy in his postoperative computed tomography scan. The possible relationship of this finding to the size of the hematoma and the outcome is discussed.

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.

[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.