Increased inherent intestinal granzyme B expression may be associated with SIV pathogenesis in Asian non-human primates.

BACKGROUND: Unlike Asian non-human primates, chronically SIV-infected African non-human primates (NHP) display a non-pathogenic disease course. The different outcomes may be related to the development of an SIV-mediated breach of the intestinal mucosa in the Asian species that is absent in the African animals. METHODS: To examine possible mechanisms that could lead to the gut breach, we determined whether the colonic lamina propria (LP) of SIV-naïve Asian monkeys contained more granzyme B (GrB) producing CD4 T cells than did that of the African species. GrB is a serine protease that may disrupt mucosal integrity by damaging tight junction proteins. RESULTS: We found that the colonic LP of Asian NHP contain more CD4(+) /GrB(+) cells than African NHP. We also observed reduced CD4 expression on LP T cells in African green monkeys. CONCLUSION: Both phenotypic differences could protect against SIV-mediated damage to the intestinal mucosa and could lead to future therapies in HIV(+) humans.

Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex.

A tetrameric recombinant major histocompatibility complex (MHC) class I-peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and beta2-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C-M) representing the optimal nine-amino acid peptide of Mamu-A*01-restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C-M complex. Tetrameric Mamu-A*01/p11C, C-M complex bound to T cells of SIVmac-infected, Mamu-A*01(+), but not uninfected, Mamu-A*01(+), or infected, Mamu-A*01(-) rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01(+) rhesus monkeys was only found in the cluster of differentiation (CD)8alpha/beta+ T lymphocyte subset and the percentage of CD8alpha/beta+ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01+ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C-M complex-binding, but not nonbinding, CD8alpha/beta+ T cells. Furthermore, the percentage of CD8alpha/beta+ T cells binding this tetrameric Mamu-A*01/p11C, C-M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings.

Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes.

Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.

Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.

Efficient entry inhibition of human and nonhuman primate immunodeficiency virus by cell surface-expressed gp41-derived peptides.

Membrane-anchored C-peptides (for example, maC46) derived from human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 effectively inhibit HIV-1 entry in cell lines and primary human CD4+ cells in vitro. Here we evaluated this gene therapy approach in animal models of AIDS. We adapted the HIV gp41-derived maC46 vector construct for use in rhesus monkeys. Simian immunodeficiency virus (SIV and SHIV) sequence-adapted maC46 peptides, and the original HIV-1-derived maC46 expressed on the surface of established cell lines blocked entry of HIV-1, SIVmac251 and SHIV89.6P. Furthermore, primary rhesus monkey CD4+ T cells expressing HIV sequence-based maC46 peptides were also protected from SIV entry. Depletion of CD8+ T cells from PBMCs enhanced the yield of maC46-transduced CD4+ T cells. Supplementation with interleukin-2 (IL-2) increased transduction efficiency, whereas IL-7 and/or IL-15 provided no additional benefit. Phenotypic analysis showed that maC46-transduced and expanded cells were predominantly central memory CD4+ T cells that expressed low levels of CCR5 and slightly elevated levels of CD62L, beta7-integrin and CXCR4. These findings show that maC46-based cell surface-expressed peptides can efficiently inhibit primate immunodeficiency virus infection, and therefore serve as the basis for evaluation of this gene therapy approach in an animal model for AIDS.

[Amiodarone-induced pneumonitis. Lethal complication in a patient after thoracic surgery]. / Amiodaroninduzierte Pneumonitis. Letale Komplikation bei einem thoraxchirurgischen Patienten.

The case of a 69-year-old patient is reported with non-small cell lung cancer who underwent lobectomy with sleeve resection and postoperatively developed intermittent atrial tachyarrhythmia. The patient was treated with a beta-blocker and the class III antiarrhythmic drug amiodarone, which resulted in normal frequency sinus rhythm. After pharmacological saturation with amiodarone, fibrosis of the lungs developed with subsequent respiratory insufficiency. Despite maximum intensive care therapy the patient died 9 weeks after surgery of hypoxemia-related multiple organ dysfunction syndrome. Based on this case amiodarone-related pneumonitis will be discussed, which due to a lack of pathognomonic symptoms is often difficult to diagnose and for which there are few treatment options available. The differential diagnoses of pneumonitis will also be discussed.

Detection of viral RNA in CD4(-)CD8(-) and CD4(-)CD8(+) lymphocytes in vivo in rhesus monkeys infected with simian immunodeficiency virus of macaques.

A definition of the specific cell types that support HIV replication early in the course of infection will be important for understanding AIDS pathogenesis and designing strategies for preventing infection. Observations have indicated that the population of lymphocytes susceptible to productive infection extends beyond activated CD4(+) T cells. To explore this issue, we have employed laser scanning cytometry technology and the techniques of lymphocyte surface immunophenotyping followed by fluorescent in situ hybridization to detect simian immunodeficiency virus of macaques (SIVmac) RNA in phenotypically defined rhesus monkey lymphocytes. The immunophenotype of productively infected cells in either a rhesus monkey T cell line or in PBMCs infected in vitro with SIVmac was remarkably similar to that observed in productively infected PBMCs obtained from monkeys during primary infection. We observed low levels or no detectable expression of CD4 on cells infected in vitro or on PBMCs of infected monkeys. However, a substantial number of SIVmac-infected PBMCs both in cultured lymphocytes and sampled directly from infected monkeys expressed CD8 but not CD4. These observations are consistent with the possibility that the CD4 molecule may be modulated off the surface of CD4(+)CD8(-) or CD4(+)CD8(+) lymphocytes after infection or that infection occurred via a CD4-independent mechanism. Moreover, there was no preferential expression of CD25 on cells positive for SIVmac RNA, which might have been predicted if replication of the virus was occurring selectively in activated lymphocytes. These results broaden the range of lymphocytes that support productive SIVmac infection to include CD4(-)CD8(-) and CD4(-)CD8(+) subsets, and are consistent with virus replication occurring in nonactivated cells.

Effect of complement consumption by cobra venom factor on the course of primary infection with simian immunodeficiency virus in rhesus monkeys.

Cobra venom factor (CVF)-induced consumption of complement proteins was used to investigate the role of complement in vivo in the immunopathogenesis of simian immunodeficiency virus of macaques (SIVmac) infection in rhesus monkeys. Repeated administration of CVF was shown to deplete complement to <5% of baseline hemolytic activity of serum complement for 10 days in a normal monkey. Three groups of SIVmac-infected animals were then evaluated: monkeys treated with CVF resulting in complement depletion from days -1 to 10 postinfection, monkeys treated with CVF resulting in complement depletion from days 10 to 21 postinfection, and control monkeys that received no CVF. CD8+ SIVmac-specific cytotoxic T lymphocyte (CTL) generation and CD4+ T lymphocyte depletion during primary infection were not affected by CVF treatment. Viral load, assessed by measurements of plasma p27gag antigen and viral RNA, was transiently higher during the first 4 weeks following infection in the CVF-treated monkeys and the subsequent clinical course in these treated animals was accelerated. These results suggest that complement proteins may participate in immune defense mechanisms that decrease virus replication following the initial burst of intense viremia during primary SIVmac infection. However, we cannot rule out that the observed increased virus replication was induced by immune activation resulting from the administration of a foreign antigen to these monkeys.

An IL-2/Ig fusion protein influences CD4+ T lymphocytes in naive and simian immunodeficiency virus-infected Rhesus monkeys.

The T cell-stimulatory cytokine interleukin 2 (IL-2) is being evaluated as a therapeutic in the clinical settings of HIV infection and cancer. However, the clinical utility of IL-2 may be mitigated by its short in vivo half-life, toxic effects, and high production costs. We show here that an IL-2/Ig fusion protein possesses IL-2 immunostimulatory activity in vitro and a long in vivo half-life. IL-2/Ig treatment of healthy rhesus monkeys induced significant increases in CD4(+) T lymphocyte counts and expression of CD25 by these cells. Short courses of IL-2/Ig treatment of simian immunodeficiency virus (SIV)-infected rhesus monkeys in conjunction with antiretroviral drugs resulted in increased CD25 expression on T lymphocytes, and transient increases in CD4(+) T lymphocyte counts. Plasma viremia did not increase in these treated animals. Treatment of healthy or SIV-infected rhesus monkeys with a plasmid encoding the IL-2/Ig protein did not affect CD4(+) T lymphocytes. These results demonstrate that IL-2/Ig has potential utility as an immunostimulatory therapeutic.

Influence of continuous haemofiltration on haemodynamics and central blood volume in experimental endotoxic shock.

In order to assess the influence of continuous haemofiltration (HF) on haemodynamics and central blood volume in endotoxic shock, endotoxinaemia was invoked in 20 swine (28-32 kg). 15 min after doubling the mean pulmonary pressure, the animals were randomly assigned to receive either a zero-balanced veno-venous HF with an ultrafiltration and replacement rate of 600 ml/h (HF group, n = 10) or to observe the spontaneous course (E group, n = 10) under a constant infusion of endotoxin for 4 h. A trend to a higher survival rate in the HF group (6/10 vs. 3/10; E group) during the observation period was evident, but not statistically significant. Early initiation of HF during endotoxic shock modifies the haemodynamic response, lowering the pulmonary artery pressure (PAP), PCWP, pulmonary (PVR) and systemic vascular resistance (SVR), compared to the spontaneous course, whereas the decrement of central blood volume was comparable in both groups. These changes cannot be explained by effects of the HF on the volume status, but supports and additional effect by the filtration of small and medium-sized molecules.

The consequences of continuous haemofiltration on lung mechanics and extravascular lung water in a porcine endotoxic shock model.

Endotoxinaemia (E. coli endotoxin, 0.111.B4) and pulmonary hypertension were evoked in 20 swine, randomly assigned to receive either zero-balanced venovenous haemofiltration (HF) with an ultrafiltration and replacement rate of 600 ml/h (HF group, n = 10) or to undergo an uninfluenced spontaneous course (E group, n = 10) during a constant infusion of endotoxin until the end of the experiment. Endotoxin-induced pulmonary dysfunction was assessed on the basis of extravascular lung water (EVLW) using a thermo-dye technique via a fiberoptic intra-aortic probe, gas exchange and lung mechanics, the latter derived by a pressure-volume loop (P/V loop) of the respiratory system (super syringe, flow 30 ml/s, tidal volume 600 ml). A comparable increase in alveolo-arterial oxygen difference and a constant EVLW was observed in both groups. The progressive deterioration of hysteresis area and compliance parameters by endotoxinaemia was significantly blunted by HF. Independent of an impact on pulmonary oedema zero-balanced HF modifies endotoxin induced lung injury, probably by the convective transport of mediator substances.

The effect of solutions of varying branched-chain concentration on the plasma amino acid pattern and metabolism in intensive care patients.

30 intensive care surgical patients were investigated over a period of five days following trauma or major surgery. They were randomised by card into two groups, Group I received an amino acid solution containing 45 per cent branched-chain amino acids (BCAA), and group II an amino acid solution with a 10 per cent content of BCAAs. After 24 hours of infusion, the total amino acid concentration and branched-chain amino acid concentration in the plasma of Group I patients already clearly exceeded the normal range, and continued to increase during the period of study. In Group II these parameters rapidly returned to normal where they remained. Cumulative negative nitrogen balance and nitrogen excretion on each day of investigation were significantly less in Group II. These results indicate that after severe trauma or major surgery amino acid solutions containing high concentrations of BCAAs may be an unphysiological load on the already stressed metabolism, rather than a benefit.

Estimating mineral changes in enamel formation by ashing/BSE and microCT.

Enamel formation produces the most highly mineralized tissue in the human body. The growth of enamel crystallites is assisted by enamel proteins and proteinases. As enamel formation progresses from secretory to maturation stages, the composition of the matrix with its mineral and non-mineral components dynamically changes in an inverse fashion. We hypothesized that appropriately calibrated micro-computed tomography (µCT) technology is suitable to estimate the mineral content (weight and/or density) and volume comparable in accuracy with that for directly weighed and sectioned enamel. Different sets of mouse mandibular incisors of C57BL/6 mice were used for dissections and µCT reconstructions. Calibration phantoms corresponding to the range of enamel mineral densities were used. Secretory-stage enamel contained little mineral and was consequently too poor in contrast for enamel volumes to be accurately estimated by µCT. Maturation-stage enamel, however, showed remarkable correspondence for total mineral content per volume where comparisons were possible between and among the different analytical techniques used. The main advantages of the µCT approach are that it is non-destructive, time-efficient, and can monitor changes in mineral content of the most mature enamel, which is too physically hard to dissect away from the tooth.

Type 2 diabetes is associated with vertebral fractures in a sample of clinic- and hospital-based Latinos.

Latinos are the fastest growing ethnic population in the United States and type 2 diabetes is a major health burden in this population, but little effort has been made to study the prevalence of diabetic vertebral fragility in Latinos. We performed a cross-sectional study to determine vertebral fracture prevalence in a hospital-based population of South Texas residents (N = 296). We defined fractures in X-rays as a >20% reduction in vertebral body height. Numerous variables were recorded, including age, body mass index, indicators of diabetes management and others. 71% of the sample (N = 296) was Latino. The prevalence of vertebral fracture was increased in diabetic subjects relative to non-diabetic subjects (diabetic 27.9%, non-diabetic 13.8%) and, regardless of sex and diabetics status, decreased in Latinos relative to non-Latinos (Latino 16.7%, non-Latino 26.4%). These data suggest that vertebral fractures may be a growing concern for diabetic Latinos as well as diabetics of any racial/ethnic background.

[A special nutrition case: the infected patient]. / Der besondere Ernährungsfall: der septische Patient.

Severe septicemia is commonly a catabolic disease process with increased energy demands and enhanced protein degradation. Septic ICU-patients are on the one hand dependent on a sufficient substrate application; on the other hand, however, the organism's tolerance against exogenous substrate application is very often diminished in these patients because of varying organ insufficiencies. Because septicemia is not a uniform type of illness with predictable organ dysfunctions, it is not possible to give recommendations for a specific nutritional diet in septic patients. Nutritional management must be adapted individually according to the type and the degree of organ dysfunctions associated with septicemia.

[Effect of metabolism-oriented substrate administration on energy and protein metabolism in polytraumatized artificial respiration patients]. / Untersuchungen über den Einfluss einer umsatzorientierten Substratzufuhr auf den Energie- und Proteinstoffwechsel polytraumatisierter Beatmungspatienten.

An adequate "individually tailored" infusion and nutritional therapy is one of the essential prerequisites for an optimal healing process - especially in ventilated, polytraumatized patients with reduced compensatory capacities. There are nevertheless practically no publications dealing with the effect of substrate application adjusted to the measured metabolic rate on the energy and protein metabolism of the critically ill. In order to clarify this situation a prospective study was carried out on a group of 40 polytraumatized, ventilated patients, who were randomized into four groups, each receiving different infusion and nutritional regimen. The O2-consumption, energy expenditure, nitrogen balance and substrate concentrations in plasma and urine were determined, and the urea production rate and substrate turnover of all patients calculated. In the groups given nutritional support carbohydrate application adjusted to O2-consumption - lead to blood glucose concentrations which were persistently high. However, median values did not exceed 10 mmol/l and insulin application was never necessary. Energy expenditure - calculated from O2-consumption - averaged about 3000 kcal/day and was clearly below values previously reported in the literature for patients comparable to those studied in this investigation. There was no difference in energy expenditure between the patients treated with various infusion regimen. In none of the groups the median plasma urea concentration did exceed reference range. Despite an apparent improvement in nitrogen retention rate - through an increased amino-acid intake and a balanced energy input - an increased urea production rate resulted. When a balanced delivery of energy-yielding substrates is given, 2 g amino-acids/kg/day seems to be the upper limit of nitrogen support in the critically ill. 3-methylhistidine excretion in urine was parallel to urea production rate, indicating that the amino-acid sparing effect of carbohydrates is mainly derived from amino-acid conservation in muscle. These results seem to indicate that even in the early posttraumatic period a substrate application, adjusted to the measured turnover is possible without leading to a disturbance in homeostasis.

[Particles in infusion solutions]. / Partikel in Infusionslösungen.

The significance of infusion therapy has increased considerably in the last two decades because of a constant expansion of indications and therapeutic possibilities. Like all other types of therapy, the efficiency of infusion therapy depends on the care taken in determining the indication and in administration. It is therefore imperative that everybody directly involved in this type of therapy is informed of the problems connected with the use of infusion solutions and aware of the specific risks of the infusion techniques in use. One essential risk inherent in the use of infusion solutions is the hazard of particulate contamination. This paper describes the potential sources which can lead to an influx of particles into the organism. Furthermore it is demonstrated that nowadays the primary or intrinsic contamination rate of commercially available solutions during manufacture and storage--as a result of the stringent regulations of the GMP (good manufacturing practice) is far below the high standard of the Australian pharmacopoea. The in-use or extrinsic contamination of infusion solutions, however, must be taken seriously as shown by the sharp increase of particulate contamination during routine hospital work. Under this aspect contamination of the organism with particles particularly increases because of additional injections into the tubing of the infusion system. Besides reducing additional administration of drugs into the infusion bottle or the infusion line to an absolutely necessary minimum, 5-micron filters have been found to effect a more than 90% reduction of particle influx as well as being easy to handle during routine clinical work.