High levels of CD34+CD38low/-CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study

Background

Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34⁺CD38^low/− compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34⁺CD38^low/−CD123⁺ phenotype.

Design and Methods

The percentage of CD34⁺CD38^low/−CD123⁺ cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses.

Results

A proportion of CD34⁺CD38^low/−CD123⁺ cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34⁺CD38^low/−CD123⁺ higher than 15% retained significance with an odds ratio of 0.33 (0.1–0.97; P=0.044). A greater than 1% population of CD34⁺CD38^low/−CD123⁺ cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34⁺CD38^low/−CD123⁺ cells retained prognostic significance for both parameters after multivariate analysis.

Conclusions

The percentage of CD34⁺CD38^low/−CD123⁺ leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure.     

Blastic plasmacytoid dendritic cell neoplasm in children: diagnostic features and clinical implications

Background

Blastic plasmacytoid dendritic cell neoplasm is a rare malignancy that typically follows a highly aggressive clinical course in adults, whereas experience in children with this disease is very limited.

Design and Methods

This retrospective study analyzed the pathological and clinical findings of nine cases of blastic plasmactyoid dendritic cell neoplasm presenting in patients under the age of 18 years who were reviewed at our institution. We also identified 20 well-documented additional pediatric cases in the literature.

Results

In the combined analysis, the overall survival rate among the 25 patients with available follow-up, all having received chemotherapy, was 72% (follow-up ranging from 9 months to 13 years, with a median of 30 months). The event-free survival rate was 64%. Nine patients were alive 5 years after the original diagnosis, although only three of them had undergone hematopoietic stem cell transplantation – one in first complete remission and two in second remission. Of the seven patients who lacked cutaneous disease at presentation, 100% survived, including five who were alive more than 5 years after diagnosis, although only two had undergone stem cell transplantation. Among the 18 patients who presented with cutaneous disease and for whom follow-up data were available, only 11 survived (61%). Detailed immunophenotypic characterization and clinical features of all cases are presented. Unexpectedly, three of four cases of blastic plasmacytoid dendritic cell neoplasm tested showed focal positivity for S-100. S-100 was negative in 28 cases of acute myeloid leukemia evaluated for this marker.

Conclusions

In contrast to adult cases, in which long-term survival depends on stem cell transplantation in first complete remission, blastic plasmacytoid dendritic cell neoplasms in children are clinically less aggressive. Treatment with high-risk acute lymphoblastic leukemia-type chemotherapy appears to be effective, and stem cell transplantation may be reserved for children who relapse and achieve a second remission. Outcomes were more favorable in cases that lacked cutaneous disease at presentation, although a comparison of cutaneous and non-cutaneous cases might be confounded by differences in treatment regimens. Focal expression of S-100 may be seen in concert with other markers of plasmacytoid dendritic cells.     

The Role of Immune Cells in Chronic HBV Infection

Hepatitis B virus (HBV) infection is a major cause of chronic liver diseases that may progress to liver cirrhosis and hepatocellular carcinoma. Host immune responses are important factors that determine whether HBV infection is cleared or persists. After infection, viral replication occurs inside hepatocytes, and the secretion of infectious virions can take place at high rates for decades. Consequently, HBV DNA and viral proteins, like HBV early antigen (HBeAg) and HBV surface antigen (HBsAg), can be easily detected in serum. Chronic infection with HBV is the result of an ineffective antiviral immune response towards the virus. In this review, we discuss the role of immune cells in chronic HBV infection.     

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

Background

Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky.

Design and Methods

Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient’s erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient’s erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed.

Results

In the cold, the cryohydrocytosis patient’s erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO₃-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na⁺/K⁺ exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K⁺ uptake. Unidirectional K⁺ influx measurements showed that the patient’s cells have abnormally high activities of the cation-proton exchanger and the K⁺,Cl⁻ co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient’s erythrocytes differed from that of healthy donors.

Conclusions

These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.     

Peripheral killer cells do not differentiate between asthma patients with or without fixed airway obstruction

Objective: The three main types of killer cells – CD8⁺ T cells, NK cells and NKT cells – have been linked to asthma and chronic obstructive pulmonary disease (COPD). However, their role in a small subset of asthma patients displaying fixed airway obstruction (FAO), similar to that seen in COPD, has not been explored. The objective of the present study was to investigate killer cell numbers, phenotype and function in peripheral blood from asthma patients with FAO, asthma patients without FAO, and healthy individuals. Methods: Peripheral CD8⁺ T cells (CD8⁺CD3⁺CD56^?), NK cells (CD56⁺CD3^?) and NKT-like cells (CD56⁺CD3⁺) of 14 asthma patients with FAO (post-bronchodilator FEV/FVC <0.7, despite clinician-optimised treatment), 7 asthma patients without FAO (post-bronchodilator FEV/FVC ≥ 0.7), and 9 healthy individuals were studied. Results: No significant differences were seen between the number, receptor expression, MAPK signalling molecule expression, cytotoxic mediator expression, and functional cytotoxicity of peripheral killer cells from asthma patients with FAO, asthma patients without FAO and healthy individuals. Conclusions: Peripheral killer cell numbers or functions do not differentiate between asthma patients with or without fixed airway obstruction.     

Effects of baicalin in CD4 + CD29 + T cell subsets of ulcerative colitis patients

AIM: To evaluate the role of baicalin in ulcerative colitis (UC) with regard to the CD4⁺CD29⁺ T helper cell, its surface markers and serum inflammatory cytokines.METHODS: Flow cytometry was used to detect the percentage of CD4⁺CD29⁺ cells in patients with UC. Real time polymerase chain reaction was used to detect expression of GATA-3, forkhead box P3, T-box expressed in T cells (T-bet), and retinoic acid-related orphan nuclear hormone receptor C (RORC). Western blotting was used to analyze expression of nuclear factor-κB (NF-κB) p65, phosphorylation of NF-κB (p-NF-κB) p65, STAT4, p-STAT4, STAT6 and p-STAT6. The concentrations of interferon-γ (IFN-γ), interleukin (IL)-4, IL-5, IL-6, IL-10 and TGF-β in serum were determined by ELISA assay.RESULTS: The percentages of CD4⁺CD29⁺ T cells were lower in treatment with 40 and 20 μmol/L baicalin than in the treatment of no baicalin. Treatment with 40 or 20 μmol/L baicalin significantly upregulated expression of IL-4, TGF-β1 and IL-10, increased p-STAT6/STAT6 ratio, but downregulated expression of IFN-γ, IL-5, IL-6, RORC, Foxp3 and T-bet, and decreased ratios of T-bet/GATA-3, p-STAT4/STAT4 and p-NF-κB/NF-κB compared to the treatment of no baicalin.CONCLUSION: The results indicate that baicalin regulates immune balance and relieves the ulcerative colitis-induced inflammation reaction by promoting proliferation of CD4⁺CD29⁺ cells and modulating immunosuppressive pathways.     

The Assessment of NK Cytotoxicity and CD56+/CD16+ Phenotype by Flowcytometry in PBL Isolated from Women with Recurrent Spontaneous Abortion

Background: Human peripheral blood NK cells constitutively express CD56 and CD16 antigens. Peripheral blood NK cells seem to be strongly related with decidual NK cells, and may reflect the decidual NK cell functional status. There are varied reports concerning the relationship between NK cell cytotoxicity in women with recurrent spontaneous abortion. Objective: To study NK activity in women with history of RSA by using a non-radioactive cytotoxicity assay. Methods: Peripheral blood lymphocytes from RSA and healthy multiparous women were obtained. Lymphocytes were isolated and mixed with K562 NK-sensitive cell line. A non-radioactive method for NK cell cytotoxicity assessment was utilized. Dead K562 cell populations were detected by FACS Calibur flow cytometry, and the data obtained was analysed on cell-Quest software. The proportion of CD16+ /CD56+ cells was then calculated. Results: The proportion of NK cells were 9.21% ± 3.06 and 13.48% ± 4.09 in healthy women and RSA, respectively. The percentage of cytotoxicity was determined to be 19.3% ± 3.9 in healthy group and 27.1% ± 6.5 in RSA group with an effector:target ratio of 50:1. The data shows an increase in PBLs potential for in vitro cytotoxicity assay in RSA individuals. The analyses indicate that there is a weak positive correlation between NK cytotoxicity potential and the percentage of NK cells in PBL population. Conclusion: The present study demonstrates that the percentage of CD56+ /CD16+ cells increases in individuals with recurrent spontaneous abortion. We conclude that NK cytotoxicity as well as NK number is partially involved in RSA.     

Impact of constitutional polymorphisms in VCAM1 and CD44 on CD34+ cell collection yield after administration of granulocyte colony-stimulating factor to healthy donors

Background

The number of CD34⁺ cells mobilized from bone marrow to peripheral blood after administration of granulocyte colony-stimulating factor varies greatly among healthy donors. This fact might be explained, at least in part, by constitutional differences in genes involved in the interactions tethering CD34⁺ cells to the bone marrow.

Design and Methods

We analyzed genetic characteristics associated with CD34⁺ cell mobilization in 112 healthy individuals receiving granulocyte colony-stimulating factor (filgrastim; 10 μg/kg; 5 days).

Results

Genetic variants in VCAM1 and in CD44 were associated with the number of CD34⁺ cells in peripheral blood after granulocyte colony-stimulating factor administration (P=0.02 and P=0.04, respectively), with the quantity of CD34⁺ cells ×10⁶/kg of donor (4.6 versus 6.3; P<0.001 and 7 versus 5.6; P=0.025, respectively), and with total CD34⁺ cells ×10⁶ (355 versus 495; P=0.002 and 522 versus 422; P=0.012, respectively) in the first apheresis. Of note, granulocyte colony-stimulating factor administration was associated with complete disappearance of VCAM1 mRNA expression in peripheral blood. Moreover, genetic variants in granulocyte colony-stimulating factor receptor (CSF3R) and in CXCL12 were associated with a lower and higher number of granulocyte colony-stimulating factor-mobilized CD34⁺ cells/μL in peripheral blood (81 versus 106; P=0.002 and 165 versus 98; P=0.02, respectively) and a genetic variant in CXCR4 was associated with a lower quantity of CD34⁺ cells ×10⁶/kg of donor and total CD34⁺ cells ×10⁶ (5.3 versus 6.7; P=0.02 and 399 versus 533; P=0.01, respectively).

Conclusions

In conclusion, genetic variability in molecules involved in migration and homing of CD34⁺ cells influences the degree of mobilization of these cells.     

CD16+ CD56- NK cells in the peripheral blood of cord blood transplant recipients: a unique subset of NK cells possibly associated with graft-versus-leukemia effect

A marked increase in CD16+ CD56- NK cells in the peripheral blood (PB) was observed in a cord blood transplant (CBT) recipient with refractory acute myeloid leukaemia (AML) in association with attaining molecular remission. CD16+ CD56- NK cells isolated from the patient became CD16+CD56+NKG2D+ when they were cultured in the presence of IL-2. Although cultured CD16+CD56- NK cells retained the killer-cell immunoglobulin receptor (KIR)-ligand (KIR-L) specificity and the patient's leukemic cells expressed corresponding KIR ligands, they killed patient's leukemic cells expressing ULBP2. The cytotoxicity by cultured CD16+CD56- NK cells was abrogated by anti-ULBP2 antibodies. When leukemic cells obtained at relapse after CBT were examined, both the ULBP2 expression and susceptibility to the cultured NK cells decreased in comparison to leukemic cells obtained before CBT. An increase in the CD16+CD56- NK cell count (0.5 x 10(9)/L or more) in PB was observed in seven of 11 (64%) CBT recipients but in none of 13 bone marrow (BM) and eight peripheral blood stem cell (PBSC) transplant recipients examined during the similar period after transplantation. These findings suggest an increase in CD16+CD56- NK cells to be a phenomenon unique to CBT recipients and that mature NK cells derived from this NK cell subset may contribute to the killing of leukemic cells expressing NKG2D ligands in vivo.     

Changes in bone marrow and peripheral blood lymphocyte subset findings with onset of hepatitis-associated aplastic anemia

Rationale:Hepatitis-associated aplastic anemia (HAAA) is a rare illness that results in bone marrow failure following hepatitis development. The etiological agent remains unknown in most HAAA cases. However, clinical features of the disease and immunotherapy response indicate that immune-mediated factors play a central role in the pathogenesis of HAAA. Activation of cytotoxic T cells and increase in CD8 cells could exert cytotoxic effects on the myelopoietic cells in the bone marrow.Patient concerns:A 15-month-old boy was brought to our hospital with complaints of generalized petechiae and purpura observed a week prior to hospitalization. His liver was palpated 3 cm below the costal margin, platelet count was 0 × 10⁴/μL, and alanine aminotransferase level was 1346 IU/L. A blood test indicated cytomegalovirus infection, and 3 bone marrow examinations revealed progressive HAAA. As the disease progressed to the 3^rd, 6^th, and 9^th week after onset, CD4⁺ T cells were markedly decreased, CD8⁺ T cells were markedly increased, and the CD4/CD8 ratio was significantly decreased. The number of B cells and natural killer cells decreased with time, eventually reaching 0.0%.Diagnosis:HAAA.Interventions:Rabbit antithymocyte globulin and eltrombopag olamine (a thrombopoietin receptor agonist) were administered.Outcomes:The patient''s platelet count returned to normal, and bone marrow transplantation was avoided. The peripheral blood lymphocytes (PBLs) improved as the patient''s general condition recovered.Lessons:This case demonstrates that HAAA induced by cytomegalovirus infection features decreasing CD4⁺ and increasing CD8⁺ PBLs as the bone marrow hypoplasia progresses. The PBLs return to their normal levels with the recovery from the disease. Our case findings thus support the involvement of immunological abnormality in HAAA.     

Functional analysis of synovial fluid and peripheral blood T cells from patients with rheumatoid arthritis

Rheumatoid arthritis is a T cell-mediated autoimmune disease. The lack of knowledge of the involved target antigens severely hampers research on relevant T cells in patients. Here we describe the functional analysis of freshly isolated T cells from the peripheral blood and the site of the lesion (synovial fluid or synovial membrane) of patients with rheumatoid arthritis. Healthy donors and osteoarthritis patients served as controls. Using various polyclonal stimuli, we analyzed CD4⁺ T cells with respect to proliferation and their ability to produce lymphokines. Our data show that lesion-derived CD4⁺ T cells of patients with rheumatoid arthritis are severely defective in proliferation and lymphokine (interleukin-2, interleukin-4, tumor necrosis factor-, interferon-) production. This activation defect was most pronounced at lower cell densities and was present in both synovial fluid derived and synovial membrane derived CD4⁺ T cells of all patients tested. No difference was found between responses of synovial fluid derived CD4⁺ T cells from osteoarthritis patients and those observed with peripheral blood derived T cells from all groups. The observed defect in lesion-derived CD4⁺ T cells from rheumatoid arthritis patients was not due to the effect of inflammatory factors in the synovial fluid because preincubation with synovial fluid could not induce a similar defect in control T cells. Together, our data show a rheumatoid arthritis specific, general defect in the activation of lesion-derived CD4⁺ T cells.Abbreviations IFN Interferon - IL Interleukin - MNC Mononuclear cells - OA Osteoarthritis - PB Peripheral blood - PSA Phosphate-buffered saline+bovin serum albumin+NaN₃ - PIAlb Phosphate-buffered saline+0.38% trisodiumcitrate+10% human albumin - RA Rheumatoid arthritis - SF Synovial fluid - SM Synovial membrane - TNF Tumor necrosis factor     

Identification of del(6)(q21q25) as a recurring chromosomal abnormality in putative NK cell lymphoma/leukaemia

The putative natural killer (NK) cell lymphomas/leukaemias are a group of recently characterized haematolymphoid malignancies sharing an immunophenotype of CD3/Leu4⁻ CD3ɛ⁺ CD56⁺, and a genotype of germline T-cell receptor genes. They frequently present in extranodal sites and exhibit a highly aggressive clinical course. Information on the cytogenetic or molecular events leading to the tumourigenesis in this group of tumours is very scarce. In this study we analysed the cytogenetic findings of seven patients with CD56-positive putative NK cell lymphoma/leukaemia. Three cases, including one nasal, one extranasal and one leukaemic form, showed a common region of deletion at 6q21-q25, suggesting that this may be a non-random chromosomal aberration.     

Reduced autophagy in livers of fasted,fat-depleted,ghrelin-deficient mice: Reversal by growth hormone

Plasma growth hormone (GH) and hepatic autophagy each have been reported to protect against hypoglycemia in the fasted state, but previous data have not linked the two. Here we demonstrate a connection using a mouse model of fasting in a fat-depleted state. Mice were subjected to 1 wk of 60% calorie restriction, causing them to lose nearly all body fat. They were then fasted for 23 h. During fasting, WT mice developed massive increases in plasma GH and a concomitant increase in hepatic autophagy, allowing them to maintain viable levels of blood glucose. In contrast, lethal hypoglycemia occurred in mice deficient in the GH secretagogue ghrelin as a result of knockout of the gene encoding ghrelin O-acyltransferase (GOAT), which catalyzes a required acylation of the peptide. Fasting fat-depleted Goat^−/− mice showed a blunted increase in GH and a marked decrease in hepatic autophagy. Restoration of GH by infusion during the week of calorie restriction maintained autophagy in the Goat^−/− mice and prevented lethal hypoglycemia. Acute injections of GH after 7 d of calorie restriction also restored hepatic autophagy, but failed to increase blood glucose, perhaps owing to ATP deficiency in the liver. These data indicate that GH stimulation of autophagy is necessary over the long term, but not sufficient over the short term to maintain blood glucose levels in fasted, fat-depleted mice.When animals undergo a complete fast, adaptive mechanisms maintain the supply of energy to vital organs. The classic metabolic response to an acute fast was delineated in humans more than 50 y ago, largely through the work of George Cahill and colleagues (1, 2). The initial response to fasting is glycogenolysis, which releases glucose from liver and muscle. At the same time, the liver begins to synthesize and release glucose de novo, initially using lactate, which is returned to the liver from muscle through the Cori cycle. Shortly thereafter, lipolysis is activated in adipose tissue, releasing fatty acids for combustion in muscle and for the supply of energy to fuel gluconeogenesis in the liver. Hepatic fatty acid oxidation also produces the ketone bodies acetoacetate and beta-hydroxybutyrate, which can partially replace glucose for energy in brain and muscle. This entire process is orchestrated by hormones, primarily through a drop in insulin and increases in glucagon, which activates gluconeogenesis in liver (2), and growth hormone (GH), which stimulates lipolysis in adipose tissue (3).In recent years, another source of gluconeogenic substrates in liver has been identified, namely autophagy (4, 5). Autophagy is the process by which intracellular double-membrane vesicles called autophagosomes ingest cytosolic proteins and organelles (6). Autophagosomes fuse with lysosomes, creating single-membrane bounded autolysosomes that expose the ingested contents to hydrolases that break down the ingested macromolecules. Fasting induces autophagy in liver (4). Some of the autophagic end products are combusted to produce energy, and some are used as substrates for gluconeogenesis. When autophagy is prevented through germline deletion of a required protein, animals develop profound hypoglycemia on fasting (4, 7, 8).Previous studies of fasting have been conducted in humans or animals that maintain adequate adipose tissue, allowing a continuous supply of fatty acids to liver and muscle. We know little about the maintenance of blood glucose when adipose triglycerides are exhausted and fatty acids are not available. Such a fat-depleted condition occurs in humans subjected to prolonged calorie deprivation through famine, anorexia, or other causes of cachexia (9, 10).Recently, our laboratory created an experimental model of calorie restriction in fat-depleted C57BL/6 mice (11–13). In this protocol, mice are fed daily with 40% of the chow diet that the mouse would normally consume. We call this 60% calorie restriction. The mice are fed each day at 6:00 PM. Within 1 d, they become ravenously hungry, and consume all of their food by 7:00 PM. They are then fasted for the next 23 h until they are fed once again. This cycle is repeated each day for 7–9 d. We measure blood glucose each day at 5:30 PM, after the 23-h fast and immediately before the next feeding.Over the first 4 d of this regimen, WT mice lose ∼30% of their body weight, which then stabilizes. By day 4, their body fat, as measured by NMR, constitutes <2% of body weight (11, 13). By day 5 or 6, fasting blood glucose values at 5:30 PM fall to 40–60 mg/dL, and are maintained at that level through day 8. Despite this moderate hypoglycemia, WT mice are alert and active. They have very low concentrations of free fatty acids in plasma (≤0.07 mM) and ketone bodies (≤0.12 mM). The hormonal response includes a marked decrease in insulin, increase in glucagon, and increases in ghrelin and GH, with the latter two rising progressively through 8 d (11, 13).Survival of these fasted, fat-depleted WT mice requires ghrelin, which acts by stimulating secretion of GH (11). Ghrelin, a 28-aa peptide produced primarily by enteroendocrine cells in the stomach and duodenum, acts in the arcuate nucleus of the hypothalamus and in somatotrophs in the pituitary to release GH (14–16). Ghrelin activity requires ghrelin O-acyltransferase (GOAT), which attaches octanoate, an eight-carbon fatty acid, to serine-3 of ghrelin before secretion (17, 18). Mice with germline deletions of ghrelin or Goat are unable to maintain viable levels of blood glucose when subjected to 60% calorie restriction as described above (11, 13). The calorie-restricted, ghrelin-deficient mice lose body weight and body fat as rapidly as WT mice, and become just as hungry, consuming all of their food within 60 min. The ghrelin-deficient mice are unable to stabilize their blood glucose. By day 7–8, their fasting blood glucose falls below 20 mg/dL. It rises promptly after each meal, only to fall progressively during the next 23-h fast. Profound hypoglycemia can be prevented by chronic infusion of either ghrelin or GH, begun before the onset of calorie restriction and maintained throughout (11, 13).Tracer experiments demonstrate that hypoglycemia in ghrelin-deficient, fat-depleted mice is caused by reduced production of glucose rather than by enhanced clearance (13). Plasma lactate, the major substrate for gluconeogenesis, falls in parallel with blood glucose (13). On day 7 of calorie restriction, blood glucose can be restored to WT levels by injections of pyruvate, lactate, or alanine, which provide energy and substrates for gluconeogenesis. Blood glucose is also restored by octanoate, a fatty acid that cannot be converted to glucose but can be oxidized to provide energy (13).Inasmuch as both hepatic autophagy and plasma GH are required to maintain blood glucose during fasting, the present study was designed to test the hypothesis that GH stimulates hepatic autophagy in starved, fat-depleted mice. The results demonstrate that hepatic autophagy is diminished in ghrelin-deficient mice and can be restored acutely by GH infusion. Acute restoration of hepatic autophagy does not prevent hypoglycemia, however. These results raise the possibility that GH-stimulated hepatic autophagy is necessary, but not sufficient, to maintain blood glucose in fasted, fat-depleted mice.     

Effects of aging on human leukocytes (part II): immunophenotyping of adaptive immune B and T cell subsets

Immunosenescence results from a continuous deterioration of immune responses resulting in a decreased response to vaccines. A well-described age-related alteration of the immune system is the decrease of de novo generation of T and B cells. In addition, the accumulation of memory cells and loss of diversity in antigen specificities resulting from a lifetime of exposure to pathogens has also been described. However, the effect of aging on subsets of γδTCR⁺ T cells and Tregs has been poorly described, and the efficacy of the recall response to common persistent infections in the elderly remains obscure. Here, we investigated alterations in the subpopulations of the B and T cells among 24 healthy young (aged 19–30) and 26 healthy elderly (aged 53–67) individuals. The analysis was performed by flow cytometry using freshly collected peripheral blood. γδTCR⁺ T cells were overall decreased, while CD4⁺CD8⁻ cells among γδTCR⁺ T cells were increased in the elderly. Helios⁺Foxp3⁺ and Helios⁻Foxp3⁺ Treg cells were unaffected with age. Recent thymic emigrants, based on CD31 expression, were decreased among the Helios⁺Foxp3⁺, but not the Helios⁻Foxp3⁺ cell populations. We observed a decrease in Adenovirus-specific CD4⁺ and CD8⁺ T cells and an increase in CMV-specific CD4⁺ T cells in the elderly. Similarly, INFγ⁺TNFα⁺ double-positive cells were decreased among activated T cells after Adenovirus stimulation but increased after CMV stimulation. The data presented here indicate that γδTCR⁺ T cells might stabilize B cells, and functional senescence might dominate at higher ages than those studied here.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-015-9829-2) contains supplementary material, which is available to authorized users.     

Mechanistic insights into the impairment of memory B cells and antibody production in the elderly

It is well established that immunologic memory generated early in life can be maintained into old age and mediate robust anamnestic antibody responses. Little is known, however, about the initiation of memory B cells in the elderly. We have conducted a prospective analysis of the quantities and functionalities of antigen-specific B cell responses and its association with the functional helper CD4⁺T cell responses. The ability of naïve B cells from old (60–80 years) and young (20–31 years) humans to establish functional memory was examined following primary and booster vaccination with an inactivated-virus vaccine against tick-borne encephalitis. Our data show that the number of antigen-specific memory B cells generated during primary vaccination was ～3-fold lower in old than in young individuals. The maintenance and booster responsiveness of these memory B cells were not compromised, as evidenced by similar increases in specific memory B cell frequencies upon revaccination in old and young adults. In contrast, the Ab response mediated per memory B cell after revaccination was dramatically diminished in the elderly. Also, antigen-specific IL-2-positive CD4⁺T cell responses were strongly reduced in the elderly and displayed an excellent correlation with Ab titres. The data suggest that the dramatically lower antibody response in the elderly could only partially be accounted for by the reduced B cell numbers and was strongly correlated with profound functional defects in CD4 help.     

Identification of the Genome Segments of Bluetongue Virus Type 26/Type 1 Reassortants Influencing Horizontal Transmission in a Mouse Model

Bluetongue virus serotypes 1 to 24 are transmitted primarily by infected Culicoides midges, in which they also replicate. However, “atypical” BTV serotypes (BTV-25, -26, -27 and -28) have recently been identified that do not infect and replicate in adult Culicoides, or a Culicoides derived cell line (KC cells). These atypical viruses are transmitted horizontally by direct contact between infected and susceptible hosts (primarily small ruminants) causing only mild clinical signs, although the exact transmission mechanisms involved have yet to be determined. We used reverse genetics to generate a strain of BTV-1 (BTV-1 RG_C7) which is less virulent, infecting IFNAR^(−/−) mice without killing them. Reassortant viruses were also engineered, using the BTV-1 RG_C7 genetic backbone, containing individual genome segments derived from BTV-26. These reassortant viruses were used to explore the genetic control of horizontal transmission (HT) in the IFNAR^(−/−) mouse model. Previous studies showed that genome segments 1, 2 and 3 restrict infection of Culicoides cells, along with a minor role for segment 7. The current study demonstrates that genome segments 2, 5 and 10 of BTV-26 (coding for proteins VP2, NS1 and NS3/NS3a/NS5, respectively) are individually sufficient to promote HT.