Activating KIR Haplotype Influences Clinical Outcome Following HLA-Matched Sibling Hematopoietic Stem Cell Transplantation.

Natural killer cells are thought to influence the outcome of hematopoietic stem cell transplant (HSCT), impacting on relapse, overall survival, graft versus host disease and the control of infection, in part through the complex interplay between the large and genetically diverse killer immunoglobulin-like receptor (KIR) family and their ligands. This study examined the relationship between KIR gene content and clinical outcomes including the control of opportunistic infections such as cytomegalovirus in the setting of human leucocyte antigen (HLA)-matched sibling HSCT in an Australian cohort. The presence of the KIR B haplotype which contain more activating receptors in the donor, in particular centromeric B haplotype genes (Cen-B), was associated with improved overall survival of patients with acute myeloid leukemia (AML) undergoing sibling HSCT and receiving myeloablative conditioning. Donor Cen-B haplotype was also associated with reduced acute graft versus host disease grades II-IV whereas donor telomeric-B haplotype was associated with decreased incidence of CMV reactivation. In contrast, we were not able to demonstrate a reduced rate of relapse when the donor had KIR Cen-B, however relapse with a donor Cen-A haplotype was a competing risk factor to poor overall survival. Here we show that the presence of donor activating KIR led to improved outcome for the patient, potentially through reduced relapse rates and decreased incidence of acute GvHD translating to improved overall survival. This article is protected by copyright. All rights reserved.

Quantitative description of the 3D regional mechanics of the left atrium using cardiac magnetic resonance imaging.

The left atrium (LA) plays an important role in the maintenance of hemodynamic and electrical stability of the heart. One of the conditions altering the atrial mechanical function is atrial fibrillation (AF), leading to an increased thromboembolic risk due to impaired mechanical function. Preserving the regions of the LA that contribute the greatest to atrial mechanical function during curative strategies for AF is important. The purpose of this study is to introduce a novel method of regional assessment of mechanical function of the LA. We used cardiac MRI to reconstruct the 3D geometry of the LA in nine control and nine patients with paroxysmal atrial fibrillation (PAF). Regional mechanical function of the LA in pre-defined segments of the atrium was calculated using regional ejection fraction and wall velocity. We found significantly greater mechanical function in anterior, septal and lateral segments as opposed to roof and posterior segments, as well as a significant decrease of mechanical function in the PAF group. We suggest that in order to minimize the impact of the AF treatment on global atrial mechanical function, damage related to therapeutic intervention, such as catheter ablation, in those areas should be minimized.

Trends in the use of implantable cardioverter defibrillators in Australia: a 10-year nationwide study from 2000-2009.

BACKGROUND: Implantable cardioverter defibrillators (ICD) have been demonstrated to reduce mortality in survivors of life-threatening arrhythmias (secondary prevention) and in patients at increased risk of sudden cardiac death (primary prevention). Other nations have reported significant increases in ICD use in recent years. AIM: To investigate Australian nationwide trends of ICD procedures over a 10-year period (2000-2009). METHODS: A retrospective analysis of the Australian Institute of Health and Welfare's National Hospital Morbidity Database was performed to determine the annual number of ICD implantation and replacement procedures between 2000 and 2009. Rates were calculated using Australian Bureau of Statistics data on the annual estimated population. Time trends in the yearly procedure number and rate were analysed using negative binomial regression models with comparisons made by age and sex. RESULTS: The number of new ICD implantations increased from 708 to 3198 procedures between 2000 and 2009. Replacement procedures increased from 290 to 1378. The implantation rate (per million) increased from 37.0 to 145.6 and the replacement rate from 15.1 to 62.7. When rates were adjusted for age and sex, the implantation rate increased annually by 15.8% and the replacement rate by 16.6% (P < 0.0001). Procedures occurred most commonly in men (implantations: 80.1%; replacements: 78.0%) between ages 70-79. CONCLUSIONS: ICD procedures increased significantly in Australia between 2000-2009. Despite these increases, other studies have suggested ICD devices are currently under-utilised. During the study period, males accounted for the majority of ICD procedures. While there are numerous reasons for this, it is not known if device under-use is more common in females.

Evidence-based management of heart failure in clinical practice: a review of device-based therapy use.

BACKGROUND: Heart failure is a growing health issue and is associated with significant mortality risk. Device therapy is efficacious in preventing sudden death in patients with heart failure; however, this evidence comes from rigorous clinical trials. It is unclear how device therapy is utilized in 'real-world' practice. The primary objective was to characterize patterns of device use in patients with heart failure at risk of sudden death and to identify barriers to guideline-driven prescription of implantable cardioverter-defibrillators. METHODS: We report a cross-sectional study of patients attending general cardiology clinic over a 3-month period. RESULTS: Of 1003 consecutive patients attending the cardiology clinic, 176 had heart failure. Of these, 66 were potentially eligible for device therapy, but only 16 of these had actually undergone device implantation. Potentially eligible non-recipients were older (P < 0.001), more likely to have ischaemic cardiomyopathy (P= 0.002), less likely to be prescribed spironolactone (P= 0.005) or warfarin (P= 0.02), and less likely to have a widened QRS > 120 ms (P= 0.005). There was a high prevalence of underuse of evidence-based pharmacotherapies among patients with heart failure. CONCLUSION: There is substantial underuse of device therapy in patients with heart failure. Strikingly, whereas patients with symptoms of heart failure were more likely to receive a device, those being managed for ischaemic heart disease were not. There is also a high prevalence of failure to prescribe evidence-based pharmacotherapy in a tertiary hospital general cardiology clinic. This may be explained in part by the lack of a patient database to record treatment contraindications and to alert clinicians to possible gaps in patient therapy.

The major histocompatibility complex class Ib molecule HLA-E at the interface between innate and adaptive immunity.

The non-classical major histocompatibility complex (MHC) class I molecule human leucocyte antigen (HLA)-E is the least polymorphic of all the MHC class I molecules and acts as a ligand for receptors of both the innate and the adaptive immune systems. The recognition of self-peptides complexed to HLA-E by the CD94-NKG2A receptor expressed by natural killer (NK) cells represents a crucial checkpoint for immune surveillance by NK cells. However, HLA-E can also be recognised by the T-cell receptor expressed by alphabeta CD8 T cells and therefore can play a role in the adaptive immune response to invading pathogens. The recent resolution of HLA-E in complex with both innate and adaptive ligands has provided insight into the dual role of this molecule in immunity.

CD8+ T-cell maturation following lung transplantation: the differential impact of CMV and acute rejection.

Studies on persistent viral infections demonstrate that CD8(+) T-cells differentiate along distinct pathways following chronic antigen exposure; however the effect of stimulation with non-viral chronic antigens is poorly described. We assessed the contributions that the presence of an allograft or cytomegalovirus (CMV) has on the post-thymic differentiation of CD8(+) T-cells in both the blood and lung allograft in patients undergoing lung transplantation. CD28 expression on blood CD8(+) T-cells was reduced in CMV seropositive patients, and was further reduced following acute episodes of CMV reactivation. These viral-associated changes in phenotype were not seen in CD8(+) T-cells isolated from the lung allograft where a different pattern of CD28 expression was observed. Following transplantation there was a progressive reduction in CD28 expression on BAL CD8(+) T-cells. In contrast to what was observed in peripheral blood, reduced CD28 expression on BAL CD8(+) T-cells was associated with a reduced gamma-IFN production. Furthermore, a high proportion of CD28-CD8(+) T-cells in the BAL was associated with fewer episodes of acute allograft rejection. An expanded CD28-CD8(+) T-cell subset, with reduced function, within the lung allograft may have important prognostic implications in lung transplant recipients.

Impact of indexing resting metabolic rate against fat-free mass determined by different body composition models.

OBJECTIVE: To examine the differences arising from indexing resting metabolic rate (RMR) against fat-free mass (FFM) determined using two-, three- and four-compartment body composition models. DESIGN: All RMR and body composition measurements were conducted on the same day for each subject following compliance with premeasurement protocols. SUBJECTS: Data were generated from measurements on 104 males (age 32.1+/-12.1 y (mean+/-s.d.); body mass 81.15+/-12.85 kg; height 179.5+/-6.5 cm; body fat 20.6+/-7.6%). INTERVENTIONS: Body density (BD), total body water (TBW) and bone mineral mass (BMM) were measured by hydrodensitometry, deuterium dilution and dual energy X-ray absorptiometry (DXA), respectively. These measures were used to determine two (hydrodensitometry: BD; hydrometry: TBW)-, three (BD and TBW)- and four- compartment (BD, TBW and BMM) FFM values. DXA also provided three compartment derived FFM values. RMR was measured using open circuit indirect calorimetry. RESULTS: Three (body fat group: lean, moderate, high) x five (body composition determination: hydrodensitometry, hydrometry, three-compartment, DXA, four-compartment) ANOVAs were conducted on FFM and RMR kJ.kg FFM(-1).d(-1). Within-group comparisons revealed that hydrodensitometry and DXA were associated with significant (P<0.001) overestimations and underestimations of FFM and RMR kJ.kg FFM(-1).d(-1), respectively, compared with four-compartment-derived criterion values. A significant interaction (P<0.001) resulted from DXA's greater deviations from criterion values in lean subjects. While hydrometric means were not significantly (P> or =0.68) different from criterion values intraindividual differences were large (FFM: -1.5 to 2.9 kg; RMR: -6.0 to 3.2 kJ.kg FFM(-1).d(-1)). CONCLUSION: The relationship between RMR kJ.kg FFM(-1).d(-1) and exercise status would best be investigated using three (BD, TBW)- or four (BD, TBW, BMM)-compartment body composition models to determine FFM. Other models either significantly underestimate indexed RMR (hydrodensitometry, DXA) or display large intraindividual differences (hydrometry) compared with four-compartment derived criterion values. SPONSORSHIP: Australian Research Council (small grants scheme).

Novel strategy for identification of candidate cytotoxic T-cell epitopes from human preproinsulin.

We describe a strategy for identifying ligands of human leukocyte antigen (HLA) class I molecules based on a peptide library-mediated in vitro assembly of recombinant class I molecules. We established a microscale class I assembly assay and used a capture ELISA to quantify the assembled HLA-peptide complexes. The identity of the bound ligands was then deduced by mass spectrometry. In this method, HLA complexes assembled in vitro in the presence of components of a mixture of peptides were immunoprecipitated and the bound peptide(s) identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This process of epitope extraction is robust and can be used with complex mixtures containing in excess of 300 candidate ligands. A library of overlapping peptides representing all potential octamers, nonamers and decamers from human preproinsulin was synthesized using unique library chemistry. Peptides from the library were used to initiate assembly of recombinant HLA-B8, HLA-B15 and HLA-A2, facilitating the identification of candidate T-cell epitopes from preproinsulin.

Improved rig for dynamically calibrating skinfold calipers: comparison between Harpenden and Slim Guide instruments.

This article describes an improved rig for the dynamic calibration of skinfold calipers. The new unit is 5% lighter and almost 60% smaller than its predecessor (Carlyon et al., 1996, 1998) with a 9.5 mm solid aluminium base and a quick release caliper mount providing stability to both the rig and caliper. Automation of the gap controller with an electric motor standardizes the jaw opening and closing velocity, thereby enabling hands-free operation. Frictional losses in the moving components of the rig have been reduced by replacing the main bush of the swing arm with a bearing, reducing the mass of the swing arm, adding a support wheel to the end of the swing arm, and replacing fishing swivels with a universal joint to allow for changes in the opening screw angle as the caliper's arm moves through its arc. This rig can also be adapted to different types of calipers by changing the position of the load cell, microswitches, and the caliper mount. A universal mounting bracket that can be secured to almost any table supports the rig in a vertical plane when calibrating the load cell. To demonstrate the versatility of the calibration rig, preliminary data are presented for the upscale and downscale jaw pressures of seven Harpenden and seven Slim Guide calipers.

Structure of killer cell immunoglobulin-like receptors and their recognition of the class I MHC molecules.

The recognition of class I MHC molecules by killer cell immunoglobulin-like receptors (KIR) constitutes an integral part of immune surveillance by the innate immune system. To understand the molecular basis of this recognition, the structures of several members of this superfamily have been determined. Despite their functional diversity, members of this superfamily share many conserved structural features. A central question is how these receptors recognize their ligands. The recent determination of the crystal structure of KIR2DL2 in complex with HLA-Cw3 has revealed the molecular mechanisms underpinning this interaction, which ultimately modulates the cytolytic activity of natural killer cells. While the recognition of MHC molecules by KIR is characterized by a number of unique features, some unexpected similarities with T-cell receptor recognition of MHC molecules are also observed. The detailed interactions between KIR2DL2 and HLA-Cw3 and their functional implications will be reviewed here.

Body composition changes in female bodybuilders during preparation for competition.

OBJECTIVE: To determine anthropometric and body composition changes in female bodybuilders during preparation for competition. DESIGN: There was an attempt to match subjects in the control and experimental groups for height and percentage body fat (%BF) for the initial test of this longitudinal study. SUBJECTS: Five competitive bodybuilders (-X +/- s.d.: 35.3 +/- 5.7 y; 167.3 +/- 3.7 cm; 66.38 +/- 6.30 kg; 18.3 +/- 3.5 %BF) and five athletic females (-X +/- s.d.: 30.9 +/- 13.0 y; 166.9 +/- 3.9 cm; 55.94 +/- 3.59 kg; 19.1 +/- 3.3 %BF) were recruited from advertisements in a bodybuilding newsletter and placed on sports centre noticeboards. INTERVENTIONS: The following measurements were conducted 12 weeks, 6 weeks and 3-5 d before the bodybuilders' competitions: anthropometric profile, body density by underwater weighing, total body water via deuterium dilution and bone mineral mass from a dual-energy X-ray absorptiometry scan. A combination of the last three measurements enabled the %BF to the determined by a four compartment model. RESULTS: A significant (P < or = 0.001) 5.80 kg body mass loss by the bodybuilders as they prepared for competition was primarily due to a reduction in fat mass (FM; -4.42 kg; 76.2%) as opposed to fat-free mass (FFM; -1.38 kg; 23.8%). The decreases in body mass and FM over the final 6 weeks were greater than those over the first 6 weeks. Their %BF decreased (P < 0.001) from 18.3 to 12.7, whereas the values for the control group remained essentially unchanged at 19.1-19.6 %BF. These body composition changes by the bodybuilders were accompanied by a significant decline (P < 0.001) of 25.5 mm (76.3-50.8 mm) in the sum of eight skinfold thicknesses (triceps + subscapular + biceps + iliac crest + supraspinale + abdominal + front thigh + medial calf). CONCLUSIONS: Although the bodybuilders presented with low %BFs at the start of the experiment, they still significantly decreased their body mass during the 12 week preparation for competition and most of this loss was due to a reduction in FM as opposed to FFM.

Conformational plasticity revealed by the cocrystal structure of NKG2D and its class I MHC-like ligand ULBP3.

NKG2D is known to trigger the natural killer (NK) cell lysis of various tumor and virally infected cells. In the NKG2D/ULBP3 complex, the structure of ULBP3 resembles the alpha1 and alpha2 domains of classical MHC molecules without a bound peptide. The lack of alpha3 and beta2m domains is compensated by replacing two hydrophobic patches at the underside of the class I MHC-like beta sheet floor with a group of hydrophilic and charged residues in ULBP3. NKG2D binds diagonally across the ULBP3 alpha helices, creating a complementary interface, an asymmetrical subunit orientation, and local conformational adjustments in the receptor. The interface is stabilized primarily by hydrogen bonds and hydrophobic interactions. Unlike the KIR receptors that recognize a conserved HLA region by a lock-and-key mechanism, NKG2D recognizes diverse ligands by an induced-fit mechanism.

Crystal structure of the extracellular domain of a human Fc gamma RIII.

Fc receptors play a major role in immune defenses against pathogens and in inflammatory processes. The crystal structure of a human immunoglobulin receptor, FcgammaRIIIb, has been determined to 1.8 A resolution. The overall fold consists of two immunoglobulin-like domains with an acute interdomain hinge angle of approximately 50 degrees. Trp-113, wedged between the N-terminal D1 and the C-terminal D2 domains, appears to further restrict the hinge angle. The putative Fc binding region of the receptor carries a net positive charge complementary to the negative-charged receptor binding regions on Fc. A 1:1 binding stoichiometry between the receptor and Fc was measured by both the equilibrium and nonequilibrium size-exclusion chromatography. Two separate parallel dimers are observed in the crystal lattice, offering intriguing models for receptor aggregation.

An unusual case of malignant hyperthermia during desflurane anesthesia in an African-American patient.

IMPLICATIONS: Malignant hyperthermia is an uncommon, heritable condition triggered by anesthesia and is followed by an increase in temperature that may be fatal without prompt treatment. It is rare with desflurane and in black individuals of African descent. We present a case of malignant hyperthermia in an African-American patient during desflurane anesthesia.

Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand.

Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules. Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide. KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide. No significant conformational changes in KIR occur on complex formation. The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding. Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires position 8 of the peptide to be a residue smaller than valine. A second KIR/HLA interface produced an ordered receptor-ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.

Reconstitution of bacterial expressed human CD94: the importance of the stem region for dimer formation.

Human CD94 is a subunit of the disulfide-linked, heterodimeric natural killer (NK) cell surface receptor CD94/NKG2. This receptor, a member of the C-type lectin superfamily, participates in regulating NK cell directed lysis through interaction with the major histocompatibility antigen HLA-E. Two forms of CD94 were expressed using a bacterial expression system and refolded in vitro. One form, residues 34-179, designated S34, corresponds to the entire extracellular region of the receptor, including a 23-residue stem region, and the other, residues 51-179, designated E51, corresponds only to the putative carbohydrate recognition domain of the receptor. The refolded full-length S34 protein existed as a noncovalent dimer initially but formed an interchain disulfide bond upon storage for several months. In contrast, the stemless construct, E51, existed largely as a monomeric form. The stem region of S34, residues 34-56, is sensitive to proteolysis and its absence results in dissociation of the dimer. This suggests that the residues in the stem region of CD94 help to stabilize the dimeric conformation.

Herpes simplex virus type 1-specific cytotoxic T-lymphocyte arming occurs within lymph nodes draining the site of cutaneous infection.

Various studies have shown that major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL) can be isolated from lymph nodes draining sites of cutaneous infection with herpes simplex virus type 1 (HSV-1). Invariably, detection of this cytolytic activity appeared to require some level of in vitro culture of the isolated lymph node cells, usually for 3 days, in the absence of exogenous viral antigen. This in vitro "resting" period was thought to represent the phase during which committed CD8(+) T cells become "armed" killers after leaving the lymph nodes and prior to their entry into infected tissue as effector CTL. In this study we reexamined the issue of CTL appearance in the HSV-1 immune response and found that cytolytic activity can be isolated directly from draining lymph nodes, although at levels considerably below those found after in vitro culture. By using T-cell receptor elements that represent effective markers for class I-restricted T cells specific for an immunodominant glycoprotein B (gB) determinant from HSV-1, we show that the increase in cytotoxicity apparent after in vitro culture closely mirrors the expansion of gB-specific CTL during the same period. Taken together, our results suggest that HSV-1-specific CTL priming does not appear to require any level of cytolytic machinery arming outside the lymph node compartment despite the absence of any detectable infection within that site.

Natural killer cell recognition of HLA class I molecules.

Human NK cells express multiple receptors that interact with HLA class I molecules. These receptors belong to one of two major protein superfamilies, the immunoglobulin superfamily or the C type lectin superfamily. The killer cell immunoglobulin-like receptor (KIR) family predominantly recognise classical HLA class I molecules and different family members interact with discrete HLA class I allotypes. The solution of the crystal structure of KIR2DL2 in complex with its ligand, HLA-Cw3 has provided the molecular details of a KIR/class I interaction. The interaction site spans both the alpha1 and alpha2 helices of class I and the KIR makes direct contact with peptide residues 7 and 8. The allotype specificity of KIR2DL2 for HLA-Cw3 is the result of a single hydrogen bond from Lys44 of the KIR to Asn80 of HLA-C as all other HLA-C residues that contact KIR are conserved. The lectin-like CD94/NKG2 receptors specifically interact with the non-classical class I molecule, HLA-E. Cell surface expression of HLA-E is dependent on the expression of other class I molecules as they are the major source of HLA-E binding peptides in normal cells. Consequently recognition of HLA-E by the CD94/NKG2 receptors allows NK cells to indirectly monitor the expression of a broad array of class I molecules. While the molecular interactions underlying ligand recognition by both KIR and CD94/NKG2 receptors are likely to be distinct, recognition of class I by both families of receptors appears peptide dependent. This suggest that cells that lack class I and also those that are impaired in their ability to load class I molecules with peptide will become targets for NK-mediated destruction.

Human glioma-induced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers.

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-gamma, and TNF-alpha, and increased production of IL-6 and IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression. Incubation with GCS also resulted in reduced expression of MHC class II and of CD80/86 costimulatory molecules on monocytes. The immunosuppressive effects were not the result of IL-6 or TGF-beta1 that was detected in GCS. However, it was due to a factor(s) that is resistant to pH extremes, differentially susceptible to temperature, susceptible to trypsin, and has a minimum molecular mass of 40 kDa. Our findings show that glioblastoma-generated factors that are known to suppress T cell responses alter the cytokine profiles of monocytic APC that, in turn, inhibit T cell function. This model indicates that monocytes can serve as an intermediate between tumor-generated immune-suppressive factors and the T cell responses that are suppressed in gliomas.

Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2.

Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 A, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define beta-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80 degrees, 14 degrees larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5 degrees difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface.