Sequence evolution and escape from specific immune pressure of an HIV-1 Rev epitope with extensive sequence similarity to human nucleolar protein 6.

Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8(+) T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8(+) T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4(+) T-cell count and viral load remained stable despite escape from T-cell recognition.

Inflammation-related muscle weakness and fatigue in geriatric patients.

Inflammation in older persons is associated with muscle wasting, leading to frailty and functional decline. Most studies have focused on IL-6 and TNF-α. In order to further elucidate the underlying mechanisms of muscle wasting and reduced muscle mass and strength we investigated a large panel of cytokines and chemokines, as well as cytoprotective heat shock proteins (Hsp), and measured lean body mass (LBM) and grip strength (GS), fatigue resistance (FR), and grip work (GW) in 33 geriatric patients (median age 84 years) admitted with acute infection-induced inflammation. Higher expression of Hsp27 without heat challenge (WHC) in circulating monocytes and lymphocytes correlated with better FR (r=0.363, p<0.05 and r=0.602, p<0.001 respectively) suggesting a protective effect, as Hsp27 is abundant in muscle. On the other hand, higher serum levels of the inflammatory chemokines CCL11/Eotaxin and CCL2/MCP-1 were related to lower GS and lower LBM (r=-0.393, p<0.05; r=-0.431, p<0.05) respectively. Our results point to a complex pattern of pro-and anti-inflammatory substances that interact with skeletal muscle performance during acute inflammation.

Circulating heat shock protein 70 (Hsp70) in elderly members of a rural population from Cameroon: association with infection and nutrition.

Hsp are highly conserved cytoprotective proteins which have been repeatedly portrayed at elevated levels in various infectious diseases, and there are suggestions that the presence of infectious agents may possibly be the root cause of Hsp induction. As organisms age the vulnerability to illnesses such as infection and inflammation increases and late complications due to infectious agents are mostly observed in the older part of the population. Although it is well known that environmental conditions can modulate the susceptibility to infection, and that poor nutritional status can increase the risk of contracting infection when exposed to an infectious agent, the effects of environmental conditions and nutritional status on the heat shock response have not been investigated. Therefore, we studied the heat shock response in a special elderly population living in a remote area in Cameroon, where infection and parasitosis are endemic. Our results indicate a significant increase in Hsp70 serum levels with increasing degree of inflammation. We found negative correlations between Hsp70 levels and micronutrients including vitamin D, vitamin B12, as well as folate, which could be linked to the immune modulating effects of these vitamins.

Guidelines for an integrated diagnostic approach of chronic lymphoproliferative disorders in the routine laboratory of haematology in Belgium.

This paper summarizes the minimal workout of chronic lymphoproliferative disorders in a routine laboratory of haematology as recommended by a team of experienced laboratory supervisors in Belgium, taking into account the specific organisation of healthcare in Belgium, the innovations in the field of molecular analyses and related reimbursement. The starting point was essentially based upon clinical and/or haematological indications and it is emphasized that conclusions should be drawn in close dialogue with the clinician and experts in cytogenetics and histopathology. Reports made in the laboratory should be based upon an integration of cytomorphological, immunophenotypical and molecular data. These guidelines are not intended to be used as universal 'diagnostic pathways', but should be useful in developing local diagnostic pathways. It is well understood that this consensus, being valid anno 2009, may rapidly change with new technologies being introduced and new targets discovered.

NK cell receptors and their ligands in leukemia.

Human natural killer (NK) cells are built to kill abnormal cells but to preserve autologous normal cells. To accomplish this task, they are equipped with a large number of inhibiting and activating receptors. Ligation with corresponding ligands will determine whether the NK cell becomes activated to destroy the abnormal cell. This review will focus on the abnormalities of NK cell receptors and their putative ligands found in patients with leukemia, which can lead to an inadequate function of NK cells allowing these malignant cells to escape from NK cell destruction. In recent years it has become clear that NK cells in the haploidentical hematopoietic stem cell transplantation (HSCT) setting are very effective in eliminating residual acute myeloid, but not acute lymphoid, leukemic cells. In this regard, we also reviewed published studies of retrospective cohorts of HSCT investigating the potential beneficial effect of killer-cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) ligands on NK alloreactivity. Manipulating NK cell inhibition or activation could lead to new forms of immunotherapy, ultimately leading to the elimination of resistant leukemic cells.

The effect of aging and inflammation on heat shock protein 27 in human monocytes and lymphocytes.

Heat shock proteins (Hsp) are highly conserved proteins and their synthesis is ubiquitous in virtually every species in which they have been sought. In the present study we have investigated the effect of age and inflammation on the induction of Hsp27 in human peripheral blood mononuclear cells, using flow cytometry. Sixty-six healthy control subjects or patients suffering from inflammation participated in the study. In both heat shocked (HS) and non-HS conditions, the percentage of Hsp27 producing lymphocytes as well as the intensity of Hsp27 in lymphocytes and monocytes were negatively influenced by age. The basal levels and also the levels of Hsp27 production after HS were higher for monocytes compared to lymphocytes. In addition, we found that HS resulted in a small but significant increase in the levels of Hsp27 in lymphocytes whereas a significant decrease in Hsp27 was noticed for monocytes. In conclusion, results presented herein provide evidence in support of an age-related decrease in the level of Hsp27, which disappeared in the presence of inflammation. Several relationships between the circulating levels of CRP, IL-6 and TNF-alpha with the various Hsp27 determinations were observed, indicating that cytokines are able to influence the production of Hsp27.

A defined donor activating natural killer cell receptor genotype protects against leukemic relapse after related HLA-identical hematopoietic stem cell transplantation.

Killer cell immunoglobulin-like receptors (KIRs) recognize different groups of Human Leukocyte Antigen (HLA) class I alleles and are expressed by natural killer (NK) cells and some T lymphocytes. NK cell cytotoxicity is triggered by failure to recognize the appropriate HLA class I ligand on target cells. Recently, it has been shown that HLA class I ligand incompatibility in the graft-versus-host (GvH) direction is associated with a better outcome in haploidentical hematopoietic stem cell transplantation (HSCT). Since KIR genotypes are very diverse in the population, we explored whether or not the donor KIR genotype could affect the graft-versus-leukemia (GvL) effect in the related HLA-identical HSCT setting. We determined the KIR and HLA genotypes of 65 HLA-identical patient-donor siblings. We found that the presence of two activating KIRs, 2DS1 and 2DS2, in the donor was significantly associated with a decreased leukemic relapse rate (P=0.03; OR=0.18; 95% CI: 0.037-0.88). Moreover, the probability of relapse at 5 years was significantly lower for patients who received a graft from a donor with the 2DS1(+)2DS2(+) genotype than for those who received a transplant from other donors (17 vs 63%, respectively; P=0.018). In conclusion, this study suggests that a joint effect of these two selected activating KIRs in the donor might confer some protection against leukemic relapse.

Identification of natural killer cell receptor phenotypes associated with leukemia.

Natural killer (NK) cells play a key role in defense against tumor cells that have the capacity to downregulate human leukocyte antigen (HLA) class I expression. It has been reported that leukemic cells can have downregulated expression of HLA class I molecules. The polymorphic nature of NK cell receptor (NKR) genes generates diverse repertoires in the human population, which display specificity in the innate immune response. In the present study, 11 KIR and two CD94/NKG2 receptors were genotyped by PCR-SSP in 96 leukemic patients and 148 healthy Caucasians. Here, we report a significant increased frequency of the more inhibitory AB killer cell immunoglobulin-like receptor (KIR) phenotype in leukemic patients compared to the controls (31.1% in healthy controls vs 51.0% in leukemic patients, Pc=0.002), which is related to the high prevalence of the inhibitory KIR2DL2 in this population (Pc=0.007). Moreover, two specific KIR phenotypes AB1 and AB9, including all inhibitory KIRs, were significantly associated with leukemic patients. Our study suggests that an important percentage of leukemic patients express a KIR phenotype in favor of escape from NK cell immunity.

Characterisation of pleural inflammation occurring after primary spontaneous pneumothorax.

The aim of this study was to examine the inflammatory reaction occurring in the pleural space of patients suffering from primary spontaneous pneumothorax (PSP) using pleural lavage, which was performed in patients with PSP and in healthy control subjects (essential hyperhidrosis patients undergoing thoracoscopy for sympathicolysis treatment). Cellular and solute composition of lavage fluid, peripheral blood and parietal pleural biopsies were analysed. PSP lavage fluid showed an increase in all differentiated leucocytes, but most strikingly eosinophils and neutrophils. In the blood of patients with PSP, the total number of leucocytes and the absolute number of eosinophils, neutrophils and monocytes were also significantly increased. The time in which air was present in the pleural space was positively correlated with the increase of eosinophils in lavage fluid, parietal pleura and blood. Eosinophilic cationic protein was elevated after PSP and strongly correlated with the absolute number of lavage eosinophils. Chemo and cytokine analysis in lavage fluid showed differences in concentrations of interleukin (IL)-5, IL-6, IL-8, IL-12p40, tumour necrosis factor-alpha and RANTES, but not of eotaxin. Surprisingly, high levels of lipopolysaccharide binding protein were also measured. Primary spontaneous pnumothorax is associated with a substantial pleural inflammatory reaction. The authors hypothesise that mechanical stretch factors, lipopolysaccharide binding protein/lipopolysaccharide complexes or other environmental components trigger pleural inflammation after primary spontaneous pnumothorax.

Inflammatory status as an important determinant of heat shock protein 70 serum concentrations during aging.

Heat shock proteins (Hsp) form a large family of proteins that are ubiquitously present in all organisms. In the absence of destabilising stimuli, Hsp are expressed at low levels, but their expression can be highly induced by various noxious conditions such as heat, oxygen stress and infection. Hsp have been reported to interfere with inflammatory processes and their induction is well known to decrease with aging. In the present study we have investigated Hsp 70 serum concentrations using an optimised ELISA in elderly patients, recruited from a geriatric University Hospital ward. Our results portray positive correlations between the serum levels of Hsp 70 and various markers of inflammation (monocyte count, serum concentration of TNF-alpha, plasma concentrations of C-reactive protein, and fibrinogen), explaining the difference in Hsp 70 serum concentrations in these subjects with various degrees of inflammation. We conclude that Hsp 70 is involved in inflammatory diseases and that the serum level of Hsp 70 is directly linked to the inflammatory status of the subject. However, the nature of this relationship remains to be elucidated.

Elevated serum heat-shock protein 70 levels in patients with acute infection: use of an optimized enzyme-linked immunosorbent assay.

Heat-shock proteins (Hsps) are highly conserved throughout evolution and evoke great interest both in basic biology and in medicine. They are expressed in small quantities under normal conditions, and their expression can be strongly induced by several stressors. Although their action is basically intracellular, it is now obvious that these proteins can be released into the extracellular environment from viable cells. In this study, the human Hsp 70 serum concentrations were determined using an optimized, cost-effective enzyme-linked immunosorbent assay (ELISA). The average intra-assay variation was 6%, whereas the average interassay variation was 9%. The sensitivity of the assay was 10 ng/ml, and spiking experiments showed recoveries between 101 and 109%. As an application of the technique, we have investigated the serum levels of human Hsp 70 in patients with infection and in healthy subjects. Our data show significantly higher levels of Hsp 70 (P = 0.003) in patients compared to control subjects. Positive correlations were noticed between the serum levels of Hsp 70 and various markers of inflammation (IL-6; r = 0.579, P = 0.009, TNF-alpha; r = 0.552, P = 0.012, IL-10; r = 0.361, P = 0.002). We conclude that Hsp 70 is involved in inflammation of infectious origin. The interindividual variation in the serum concentration of Hsp 70 precludes the use of serum Hsp 70 levels to distinguish patients from healthy subjects.

Proinflammatory cytokines and their role in the development of major transplant-related complications in the early phase after allogeneic bone marrow transplantation.

Serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF)-alpha were frequently measured during the first 30 days after allogeneic bone marrow transplantation (BMT) in 84 consecutive adult patients. Major transplant-related complications (MTCs) occurred in 33% of cases and included veno-occlusive liver disease, idiopathic pneumonia syndrome, severe endothelial leakage syndrome and >grade II acute graft-versus-host disease. Compared with patients having minor complications, those with MTCs developed higher levels at times of maximal clinical signs (all cytokines, P<0.001), between days 0-5 post-BMT (IL-6 and IL-8, P<0.05) and days 6-10 (L-6, P<0.001; IL-8 and TNF, P<0.01) post-BMT. We could not discriminate patterns of cytokine release that were specific for any subtype of MTC. Higher levels of IL-8 during days 0-5 were associated (P=0.044) with early (<40 days) death. Multivariate analysis including patient and transplant characteristics as well as post-BMT levels of C-reactive protein showed that high average levels of one or more of the cytokines within the first 10 days post-BMT were independently associated with MTC (Odd's ratio: 2.3 [1.2-4.5], P=0.011). This study shows that systemic release of proinflammatory cytokines contributes to the development of MTC and provides a rationale for pre-emptive anti-inflammatory treatment in selected patients.

Differential cytology of bronchoalveolar lavage fluid in asthmatic children.

Although asthma usually begins in childhood, limited information is available as to the inflammatory reaction of asthmatic children compared to adults and the influence of age. We investigated the cytology of bronchoalveolar lavage fluid (BALF) in 39 newly diagnosed wheezy children (minimum of 3 wheezing episodes during last 6 months): 21 allergic and 18 nonallergic subjects. None had received antiinflammatory treatment. Bronchoalveolar lavage (BAL) was performed, instilling 0.5 ml.kg(-1) body weight of warmed saline in 4 successive fractions. The first 2 aliquots (BALF 1) were pooled for microbiology and cytology, and the last 2 (BALF 2) for cytology only. Recovery correlated inversely with age, the most significant being for BALF 2 (r = -0.52, P = 0.001). Children under 2 years of age had larger amounts of ciliated columnar and goblet cells (P = 0.0074). Other cell types did not show age dependency. Differential cytology was characterized by a high number of creola bodies, bronchial epithelial cells (M = 68 x 10(3).ml(-1), R = 5-349), and neutrophils (M = 92 x 10(3).ml(-1), R = 0-1,257). Eosinophils were the only cells distinguishing allergic from nonallergic subjects (P = 0.003). The 16 children with positive microbiology had more neutrophils than the noninfected (P = 0.008), the latter still having more neutrophils than found in adults. These data suggest a limited age dependency in BALF cytology. Differential cytology in BALF might be helpful in differentiating asthma in children. Neutrophil inflammation might be more important than in adults.

Age-related decrease in the inducibility of heat-shock protein 70 in human peripheral blood mononuclear cells.

We have investigated the effect of age and of the presence of proinflammatory cytokines on Hsp 70 production in human peripheral blood mononuclear cells, using flow cytometry. Twenty-seven women and 23 men, all apparently healthy, participated in the study. At 37 degrees C, the percentage of Hsp 70-producing monocytes and lymphocytes, as well as the level of Hsp 70 in monocytes, were negatively influenced by age. After exposure of the cells to 42 degrees C, the increase of Hsp 70 production was more pronounced in monocytes than in lymphocytes; both the intensity of Hsp 70 production and the percentage of Hsp 70-producing cells were negatively influenced by the age of the subjects, as well for monocytes as for lymphocytes. There was a negative correlation between the intensity of Hsp 70 production by monocytes exposed to 42 degrees C and the serum levels of tumor necrosis factor-alpha and interleukin-6. In conclusion, in human monocytes and lymphocytes, heat-induced Hsp 70 production is reduced with increasing age and is negatively influenced in monocytes by proinflammatory cytokines.

The prevalence of autoantibodies in an elderly sub-Saharan African population.

In the present prospective, census-based study we have investigated the prevalence of organ-specific and non-organ-specific autoantibodies (AAb) in 152 unselected Cameroonians aged 60 years and older living in the community. AAb were detected in 49% of the participants. Non-organ-specific AAb (47%) predominated over organ-specific AAb (7%). Anti-TPO, anti-Tm, anti-Tg and anti-PC AAb were completely absent. RF was the most frequent AAb, being found in 57 (38%) cases. The prevalences of anti-SMA and RF were significantly higher in women than in men (respectively, P=0.023 and P=0.016). Higher serum concentrations of gammaglobulins were accompanied by a higher prevalence of RF (P < 0.0001) and a lower prevalence of ANA (P=0.036). The overall prevalence of AAb was higher in the filaria-infected (60%) compared to the non-infected (42%) participants (P=0.046). There was no significant influence of the vitamin D status, number of pregnancies, physical activity or medication use on the prevalence of AAb. In this study a heterogeneous pattern for the presence of the various AAb was found. Some AAb, which are commonly encountered in other studies on elderly subjects, were completely absent in this population. This diversified pattern of AAb prevalence therefore argues in favour of exogenous influences in the occurrence of AAb in elderly populations.

Soluble HLA-DR antigen levels in serum correlate with rheumatoid arthritis disease activity and the presence of disease-associated epitopes.

We investigated correlations between soluble HLA-DR (sHLA-DR) molecules and several clinical, biological and genetic parameters associated with rheumatoid arthritis (RA) disease activity. Serum sHLA-DR concentrations were determined in 146 samples from 89 RA patients by an ELISA format, using an antibody combination of mouse and rat monoclonal anti-human HLA-DR antibodies. The mean sHLA-DR serum level in RA patients was significantly increased with 277+/-19 ng/ml compared to 142+/-13 ng/ml of 80 healthy controls (P<0.001). In ascending order of significance, correlations were found between serum sHLA-DR and EULAR swelling and pain scores, Waaler-Rose, RA factor, ESR and CRP (P=0.025 to P<0.001). High sHLA-DR levels were defined above 374 ng/ml that was the 95% confidence interval of the controls. Thirty-seven blood samples (25%) in 31 RA patients were above this level. The EULAR pain and swelling scores, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and RA factor were higher (P=0.044 to P<0.001) at the moment of high sHLA-DR concentrations, compared to the lower concentrations. Higher disease activity was further found in groups of RA patients respectively heterozygous or homozygous for the disease-associated epitope (Q)R/KRAA within the HLA-DRB1 chain, compared to the group without this epitope (P<0.017 for part of the results). Likewise, sHLA-DR was respectively 169+/-17 (no disease associated epitope), 324+/-34 (heterozygous) and 442+/-69 ng/ml (homozygous for the disease-associated epitope on HLA-DRB1 alleles) (P<0.017). In conclusion, this study shows significant correlations between serum sHLA-DR levels and RA disease activity parameters, as well as increased sHLA-DR in patients with disease-associated epitope on HLA-DRB1 alleles.

HLA association of amoxicillin-clavulanate--induced hepatitis.

BACKGROUND & AIMS: Drug-induced immunoallergic hepatitis typically affects a minority of patients exposed to a particular drug. Its rarity is believed to be due to metabolic or immunologic idiosyncrasy. The presence of an immunologic idiosyncrasy might imply an HLA association. Previous studies reporting an HLA association of drug-induced hepatitis included only small numbers of patients and used serological HLA typing. METHODS: We studied 35 patients with biopsy-documented amoxicillin-clavulanate-induced hepatitis. HLA-A and -B were typed using alloantisera and compared with those of 300 controls (volunteer bone marrow donors). HLA-DRB and -DWB were typed by polymerase chain reaction-line probe assay, with 60 volunteer bone marrow donors serving as controls. RESULTS: The study group was characterized by a higher frequency of DRB1*1501-DRB5*0101-DQB1*0602 haplotype (57.1% vs. 11.7% in controls, P < 0.000005; after correction for the large number of comparisons, P < 0.0002). Patients with DRB1*1501-DRB5*0101-DQB1*0602 haplotype were more likely than patients without it to have a cholestatic (70% vs. 60%) or mixed (30% vs. 13%) than a hepatocellular pattern of hepatitis (0% vs. 27%) (P < 0.05). CONCLUSIONS: Amoxicillin-clavulanate-induced hepatitis is associated with the DRB1*1501-DRB5*0101-DQB1*0602 haplotype. The data support the view that an immunologic idiosyncrasy, mediated through HLA class II antigens, plays a role in the pathogenesis of drug-induced immunoallergic hepatitis. HLA association has a limited impact on the expression of hepatitis.

Belgian consensus recommendations for flow cytometric immunophenotyping. The Belgian Association for Cytometry/Belgische Vereniging voor Cytometrie/Association Belge de Cytométrie.

This paper summarises the guidelines and recommendations that were generated during a number of discussion forums attended by the majority of Belgian cytometry laboratory professionals. These forums focused on the rational and optimal use of flow cytometric evaluations in the clinical laboratory setting. The aim was to improve the coherence of the testing panels and the quality of the results and--as such--the clinical diagnostic information. It was also the aim to provide the Belgian prescribing physician and interested laymen with an updated overview of the flow cytometric possibilities. Emphasis is placed on immunophenotyping of haematological malignancies, hematopoietic progenitor cell counting and follow-up of the viral infection caused by the human immunodeficiency virus.

Immune dysfunction associated with chronic professional stress in nurses.

The relationship between chronic professional stress in nurses and immunity as well as the possible impact of psychopathology upon this relationship have been examined. Sixty subjects were selected on the basis of high/low scores on professional stress and psychopathology. Chronic professional stress appeared to be associated with immune dysfunction including signs of immune activation (increased numbers of cells expressing the interleukin-2 receptor, especially CD4+CD25+ cells) and possibly immune suppression (decrease in percentage of natural killer cells). The increase in activation markers, CD3+CD16CD56+ cells and serum neopterin was most pronounced in the group with high stress/low psychopathology whereas the decrease in CD8+CD11b+ cells was most pronounced in the group with high stress/high psychopathology. It is hypothesized that in the presence of chronic stress distinct psychological mechanisms are associated with specific immune dysfunctions.