S-9-PAHSA's neuroprotective effect mediated by CAIII suppresses apoptosis and oxidative stress in a mouse model of type 2 diabetes.

BACKGROUND: With the rapidly increasing prevalence of metabolic diseases such as type 2 diabetes mellitus (T2DM), neuronal complications associated with these diseases have resulted in significant burdens on healthcare systems. Meanwhile, effective therapies have remained insufficient. A novel fatty acid called S-9-PAHSA has been reported to provide metabolic benefits in T2DM by regulating glucose metabolism. However, whether S-9-PAHSA has a neuroprotective effect in mouse models of T2DM remains unclear. METHODS: This in vivo study in mice fed a high-fat diet (HFD) for 5 months used fasting blood glucose, glucose tolerance, and insulin tolerance tests to examine the effect of S-9-PAHSA on glucose metabolism. The Morris water maze test was also used to assess the impact of S-9-PAHSA on cognition in the mice, while the neuroprotective effect of S-9-PAHSA was evaluated by measuring the expression of proteins related to apoptosis and oxidative stress. In addition, an in vitro study in PC12 cells assessed apoptosis, oxidative stress, and mitochondrial membrane potential with or without CAIII knockdown to determine the role of CAIII in the neuroprotective effect of S-9-PAHSA. RESULTS: S-9-PAHSA reduced fasting blood glucose levels significantly, increased insulin sensitivity in the HFD mice and also suppressed apoptosis and oxidative stress in the cortex of the mice and PC12 cells in a diabetic setting. By suppressing oxidative stress and apoptosis, S-9-PAHSA protected both neuronal cells and microvascular endothelial cells in in vivo and in vitro diabetic environments. Interestingly, this protective effect of S-9-PAHSA was reduced significantly when CAIII was knocked down in the PC12 cells, suggesting that CAIII has a major role in the neuroprotective effect of S-9-PAHSA. However, overexpression of CAIII did not significantly enhance the protective effect of S-9-PAHSA. CONCLUSION: S-9-PAHSA mediated by CAIII has the potential to exert a neuroprotective effect by suppressing apoptosis and oxidative stress in neuronal cells exposed to diabetic conditions. Furthermore, S-9-PAHSA has the capability to reduce fasting blood glucose and LDL levels and enhance insulin sensitivity in mice fed with HFD.

Distinct Circuits From the Central Lateral Amygdala to the Ventral Part of the Bed Nucleus of Stria Terminalis Regulate Different Fear Memory.

BACKGROUND: The ability to differentiate stimuli that predict fear is critical for survival; however, the underlying molecular and circuit mechanisms remain poorly understood. METHODS: We combined transgenic mice, in vivo transsynaptic circuit-dissecting anatomical approaches, optogenetics, pharmacological methods, and electrophysiological recording to investigate the involvement of specific extended amygdala circuits in different fear memory. RESULTS: We identified the projections from central lateral amygdala (CeL) protein kinase C δ (PKCδ)-positive neurons and somatostatin (SST)-positive neurons to GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons in the ventral part of the bed nucleus of stria terminalis (vBNST). Prolonged optogenetic activation or inhibition of the PKCδCeL-vBNST pathway specifically reduced context fear memory, whereas the SSTCeL-vBNST pathway mainly reduced tone fear memory. Intriguingly, optogenetic manipulation of vBNST neurons that received the projection from PKCδCeL neurons exerted bidirectional regulation of context fear, whereas manipulation of vBNST neurons that received the projection from SSTCeL neurons could bidirectionally regulate both context and tone fear memory. We subsequently demonstrated the presence of δ and κ opioid receptor protein expression within the CeL-vBNST circuits, potentially accounting for the discrepancy between prolonged activation of GABAergic circuits and inhibition of downstream vBNST neurons. Finally, administration of an opioid receptor antagonist cocktail on the PKCδCeL-vBNST or SSTCeL-vBNST pathway successfully restored context or tone fear memory reduction induced by prolonged activation of the circuits. CONCLUSIONS: Together, these findings establish a functional role for distinct CeL-vBNST circuits in the differential regulation and appropriate maintenance of fear.

M4IDP stimulates ROS elevation through inhibition of mevalonate pathway and pentose phosphate pathway to inhibit colon cancer cells.

Maintaining redox homeostasis is an essential feature of cancer cells, and disrupting this homeostasis to cause oxidative stress and induce cell death is an important strategy in cancer therapy. M4IDP, a zoledronic acid derivative, can cause the death of human colorectal cancer cells by increasing the level of intracellular reactive oxygen species (ROS). However, its potential molecular mechanism is unclear. Our in vitro studies showed that treatment with M4IDP promoted oxidative stress in HCT116 cells, as measured by the decreased ratios of GSH/GSSG and NADPH/NADP+ and increased level of MDA. M4IDP could cause the decrease of GSH content, the increase of GSSG content, the decrease of NADPH content and pentose phosphate pathway flux, the downregulation of G6PD expression, the upregulation of unprenylated Rap1A and total expression of RhoA and CDC42. The increase of ROS and cytotoxicity induced by M4IDP could be reversed by the supplementation of NADPH, the overexpression of G6PD and the supplementation of GGOH. In vivo studies showed that M4IDP inhibited tumor growth in the human colorectal cancer xenograft mouse model, which was accompanied with a decreased [18F]FDG uptake. Collectively, these results provide evidence that M4IDP can promote oxidation in colon cancer cells by inhibiting mevalonate pathway and pentose phosphate pathway and produce therapeutic effect. This study revealed for the first time a possible mechanism of bisphosphonate-induced increase of ROS in malignant tumor cells. This is helpful for the development of new molecular therapeutic targets and can provide new ideas for the combined therapy of bisphosphonates in tumors.

Enhancement of root sulfur metabolic pathway by overexpression of OAS-TL3 to increase total soybean seed protein content.

Sulfur is essential for plant growth, and the uptake of sulfate by plant roots is the primary source of plant sulfur. Previous studies have shown that the OAS-TL gene is a key enzyme in the sulfur metabolic pathway and regulates cysteine (Cys) synthase production. However, the interaction mechanism of the glycine max OAS-TL3 Cys synthase (OAS-TL3) gene on soybean root morphology construction and seed protein accumulation is unclear. This study shows that mutant M18 has better root growth and development, higher seed protein content, and higher methionine (Met) content in sulfur-containing amino acids than wild-type JN18. By transcriptome sequencing, the differentially expressed OAS-TL3 gene was targeted in the mutant M18 root line. The relative expression of the OAS-TL3 gene in roots, stems, and leaves during the seedling, flowering, and bulking stages of the OAS-TL3 gene overexpression lines is higher than that of the recipient material. Compared to the recipient material JN74, the enzymatic activities, Cys, and GSH contents of OAS-TL are higher in the sulfur metabolic pathway of seedling roots. The receptor material JN74 is exogenously applied with different concentrations of reduced glutathione. The results demonstrate a positive correlation between reduced glutathione on total root length, projected area, surface area, root volume, total root tip number, total bifurcation number, and total crossing number. The Met and total protein contents of sulfur-containing amino acids in soybean seeds of the OAS-TL3 gene overexpression lines are higher than those of the recipient material JN74, while the gene-edited lines show the opposite results. In conclusion, the OAS-TL3 gene positively regulates soybean root growth, root activity, and the content of Met in the seeds through the OAS-TL-Cys-GSH pathway. It breaks the limitation of other amino acids and facilitates the increase of total seed protein content. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01348-y.

Distinct serotonergic pathways to the amygdala underlie separate behavioral features of anxiety.

Anxiety-like behaviors in mice include social avoidance and avoidance of bright spaces. Whether these features are distinctly regulated is unclear. We demonstrate that in mice, social and anxiogenic stimuli, respectively, increase and decrease serotonin (5-HT) levels in basal amygdala (BA). In dorsal raphe nucleus (DRN), 5-HT∩vGluT3 neurons projecting to BA parvalbumin (DRN5-HT∩vGluT3-BAPV) and pyramidal (DRN5-HT∩vGluT3-BAPyr) neurons have distinct intrinsic properties and gene expression and respond to anxiogenic and social stimuli, respectively. Activation of DRN5-HT∩vGluT3→BAPV inhibits 5-HT release via GABAB receptors on serotonergic terminals in BA, inducing social avoidance and avoidance of bright spaces. Activation of DRN5-HT∩vGluT3→BA neurons inhibits two subsets of BAPyr neurons via 5-HT1A receptors (HTR1A) and 5-HT1B receptors (HTR1B). Pharmacological inhibition of HTR1A and HTR1B in BA induces avoidance of bright spaces and social avoidance, respectively. These findings highlight the functional significance of heterogenic inputs from DRN to BA subpopulations in the regulation of separate anxiety-related behaviors.

A Deep Mesencephalic Nucleus Circuit Regulates Licking Behavior.

Licking behavior is important for water intake. The deep mesencephalic nucleus (DpMe) has been implicated in instinctive behaviors. However, whether the DpMe is involved in licking behavior and the precise neural circuit behind this behavior remains unknown. Here, we found that the activity of the DpMe decreased during water intake. Inhibition of vesicular glutamate transporter 2-positive (VGLUT2+) neurons in the DpMe resulted in increased water intake. Somatostatin-expressing (SST+), but not protein kinase C-δ-expressing (PKC-δ+), GABAergic neurons in the central amygdala (CeA) preferentially innervated DpMe VGLUT2+ neurons. The SST+ neurons in the CeA projecting to the DpMe were activated at the onset of licking behavior. Activation of these CeA SST+ GABAergic neurons, but not PKC-δ+ GABAergic neurons, projecting to the DpMe was sufficient to induce licking behavior and promote water intake. These findings redefine the roles of the DpMe and reveal a novel CeASST-DpMeVGLUT2 circuit that regulates licking behavior and promotes water intake.

The GmXTH1 gene improves drought stress resistance of soybean seedlings.

In order to study the role of GmXTH1 gene in alleviating drought stress, soybean seeds with GmXTH1 gene were transferred by T4 treated with PEG6000 concentration of 0%, 5%, 10%, and 15% respectively. The germination potential, germination rate, germination index, and other indicators were measured. The results showed that the germination potential, germination rate, and germination index of OEA1 and OEA2 strains overexpressed in T4 generation were significantly higher than those of the control material M18. After 0-day, 7-day, and 15-day drought stress, the analysis of seedling phenotypes and root-shoot of different T4 generation transgenic soybean lines showed that under stress conditions, the growth of GmXTH1 overexpression material was generally better than that of the control material M18. The growth of GmXTH1 interference expression material was generally worse than that of the control material M18, with significant differences in plant phenotypes. The root system of GmXTH1 overexpressed material was significantly developed compared with that of the control material M18. The analysis of physiological and biochemical indexes showed that the relative water content and the activity of antioxidant enzymes (superoxide dismutase and peroxidase) of GmXTH1 transgenic soybean material were significantly higher than those of the control material M18, and the accumulation of malondialdehyde was lower under the same stress conditions at seedling stage. Fluorescence quantitative PCR assay showed that the relative expression of GmXTH1 gene in transgenic soybean was significantly increased after drought stress. The results showed that the overexpression of GmXTH1 could increase the total root length, surface area, total projection area, root volume, average diameter, total cross number, and total root tip number, thereby increasing the water intake and reducing the transpiration of water content in leaves, thus reducing the accumulation of MDA and producing more protective enzymes in a more effective and prompt way, reducing cell membrane damage to improve drought resistance of soybean.

Ultrahigh activity of molybdenum/vanadium-doped Ni-Co phosphides nanoneedles based on ion-exchange for hydrogen evolution at large current density.

Heteroatom doping is a promising strategy to optimize the electronic structure of transition metal phosphides so enhancing the hydrogen evolution reaction (HER). However, complex and harsh experimental design is often required to achieve homogeneous doping of corresponding elements while achieving the best regulating effect. Herein, a facile ion-exchange (IE) strategy is applied to dope Mo/V species evenly into Ni-Co phosphides under mild conditions while maintaining the nanoneedle morphology. The electrochemical characterization verifies Mo dopants have a better electronic regulation effect on NiCoP crystal than V dopants, corresponding to the better hydrogen evolution performance of Mo-NiCoP/NF. Notably, due to the highly dispersed nanoneedle morphology, the synergistic effect of Ni-Co phosphides, and the optimized electronic structure, Mo-NiCoP/NF demonstrates a higher activity than that of the noble metal Pt/C at the high current density (>99 mA cm-2). The present work is supposed to open new sights for the development of high-performance catalysts by ion-exchange strategy.

Multicentre, prospective registry study of amyotrophic lateral sclerosis in mainland China (CHALSR): study protocol.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a representative rare disease characterised by progressive, fatal motor neuron degeneration. Due to the unknown aetiology and variability of the phenotypes, there are no accurate reports concerning the epidemiology or clinical characteristics of the disease. The low prevalence, as previously reported, makes it difficult to carry out studies with large samples. The aim of this study was to explore the natural history and clinical features of ALS in mainland China through a multicentre, prospective cohort study. The findings will both offer a better understanding of ALS and also support the development of a model to study other rare diseases. METHODS AND ANALYSIS: Patients from 88 representative hospitals in different parts of mainland China will be recruited through a specially designed online data system (http://www.chalsr.net/). We aim to recruit 4752 ALS patients over a 3-year period. Baseline data will be recorded, and follow-up data will be collected every 3 months. The primary outcome is effective survival. Overall survival and indices of disease progression will be measured as the secondary outcomes. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the ethics committee of Peking University Third Hospital (M2019388). Informed written consent will be obtained from each participant. Dissemination of the study protocol and data will take place primarily through a specially designed online data system (http://www.chalsr.net/). The collective results of the study will be published in peer-reviewed journals and shared in scientific presentations. TRIAL REGISTRATION NUMBER: NCT04328675.

Whole-Brain Map of Long-Range Monosynaptic Inputs to Different Cell Types in the Amygdala of the Mouse.

The amygdala, which is involved in various behaviors and emotions, is reported to connect with the whole brain. However, the long-range inputs of distinct cell types have not yet been defined. Here, we used a retrograde trans-synaptic rabies virus to generate a whole-brain map of inputs to the main cell types in the mouse amygdala. We identified 37 individual regions that projected to neurons expressing vesicular glutamate transporter 2, 78 regions to parvalbumin-expressing neurons, 104 regions to neurons expressing protein kinase C-δ, and 89 regions to somatostatin-expressing neurons. The amygdala received massive projections from the isocortex and striatum. Several nuclei, such as the caudate-putamen and the CA1 field of the hippocampus, exhibited input preferences to different cell types in the amygdala. Notably, we identified several novel input areas, including the substantia innominata and zona incerta. These findings provide anatomical evidence to help understand the precise connections and diverse functions of the amygdala.

MeCP2 in cholinergic interneurons of nucleus accumbens regulates fear learning.

Methyl-CpG-binding protein 2 (MeCP2) encoded by the MECP2 gene is a transcriptional regulator whose mutations cause Rett syndrome (RTT). Mecp2-deficient mice show fear regulation impairment; however, the cellular and molecular mechanisms underlying this abnormal behavior are largely uncharacterized. Here, we showed that Mecp2 gene deficiency in cholinergic interneurons of the nucleus accumbens (NAc) dramatically impaired fear learning. We further found that spontaneous activity of cholinergic interneurons in Mecp2-deficient mice decreased, mediated by enhanced inhibitory transmission via α2-containing GABAA receptors. With MeCP2 restoration, opto- and chemo-genetic activation, and RNA interference in ChAT-expressing interneurons of the NAc, impaired fear retrieval was rescued. Taken together, these results reveal a previously unknown role of MeCP2 in NAc cholinergic interneurons in fear regulation, suggesting that modulation of neurons in the NAc may ameliorate fear-related disorders.

Publisher Correction: Cannabinoid CB1 receptors in the amygdalar cholecystokinin glutamatergic afferents to nucleus accumbens modulate depressive-like behavior.

In the version of this article originally published, there were several errors in Fig. 4. In Fig. 4a, the title read '3D repeated optical inhibition after CSDS.' It should have read '3-day repeated optical inhibition after CSDS.' In Fig. 4c, two labels that should have been aligned with the time axis appeared in the wrong place in the figure. The ticks labeled 'SI' and 'Fiber implant' should have also been labeled with '10' and '14,' respectively. Additionally, in Fig. 4j, a label that should have been aligned with the time axis appeared in the wrong place in the figure. The tick labeled 'Fiber implant' should have also been labeled with '14.' The errors have been corrected in the print, PDF and HTML versions of the manuscript.

Cannabinoid CB1 receptors in the amygdalar cholecystokinin glutamatergic afferents to nucleus accumbens modulate depressive-like behavior.

Major depressive disorder is a devastating psychiatric disease that afflicts up to 17% of the world's population. Postmortem brain analyses and imaging studies of patients with depression have implicated basal lateral amygdala (BLA) dysfunction in the pathophysiology of depression. However, the circuit and molecular mechanisms through which BLA neurons modulate depressive behavior are largely uncharacterized. Here, in mice, we identified that BLA cholecystokinin (CCK) glutamatergic neurons mediated negative reinforcement via D2 medium spiny neurons (MSNs) in the nucleus accumbens (NAc) and that chronic social defeat selectively potentiated excitatory transmission of the CCKBLA-D2NAc circuit in susceptible mice via reduction of presynaptic cannabinoid type-1 receptor (CB1R). Knockdown of CB1R in the CCKBLA-D2NAc circuit elevated synaptic activity and promoted stress susceptibility. Notably, selective inhibition of the CCKBLA-D2NAc circuit or administration of synthetic cannabinoids in the NAc was sufficient to produce antidepressant-like effects. Overall, our studies reveal the circuit and molecular mechanisms of depression.

[Adenovirus-mediated PDCD5 gene transfer sensitizes apoptosis of K562 cells induced by etoposide].

This study was purposed to investigate the effect of adenovirus-mediated transfer of PDCD5 gene on apoptosis of K562 cells induced by etoposide. Recombinant adenovirus PDCD5 (Ad-PDCD5), control vectors Ad-null and Ad-eGFP were constructed by AdMax vector system respectively. After K562 cells were transfected by Ad-PDCD5, Ad-null or Ad-eGFP with different multiplicity of infection (MOI), the expression level of the PDCD5 gene was examined by RQ-RT-PCR assay. The effects of etoposide in combination with Ad-PDCD5 on the proliferation and apoptosis of K562 cells were measured by using MTT assay and flow cytometry with Annexin-V-FITC/PI dual labeling technique, respectively. The results showed that the transfection efficiencies of Ad-eGFP in K562, Jurkat and CEM cells ranged from 60% to 86%. Expression level of PDCD5 gene in K562 cells was evidently increased following transfection with Ad-PDCD5. The Ad-PDCD5 synergistically enhanced the apoptotic percentage of K562 cells induced by VP-16, as compared with that of Ad-null + VP16 and VP-16 alone respectively. It is concluded that Ad-PDCD5 may be a potential agent for enhancing the chemotherapy effect.

[Abnormal expression of PDCD5 in the bone marrow cells of adult acute myeloid leukemia].

The objective of this study was to estimate a novel apoptosis-promoting molecule PDCD5 expression in the bone marrow cells from adult acute myeloid leukemia (AML) for investigation of its significance in the pathogenesis of AML. Flow cytometry assay was used for detection of PDCD5 expression in the different groups of cells from bone marrow of AML patients and normal controls by using 21 monoclonal antibodies with different fluorescent markers. The PDCD5 expressions in bone marrow cells from some AML patients and normal controls were also detected by Western blot. The results showed that the mean PDCD5 fluorescence intensity in bone marrow nucleated cells (MNC) from the bone marrow of 36 untreated AML patients was significantly lower than that from the bone marrow of 30 normal controls (3059 +/- 1392) vs (7432 +/- 1261) (P < 0.01). The mean PDCD5 fluorescence intensity was lower in the marrow granulocytes, monocytes, blast cells, and lymphocytes from untreated AML patients than that from normal (3939 +/- 2121) vs (8367 +/- 1045); (3156 +/- 1635) vs (5917 +/- 2329); (2824 +/- 1592) vs (3998 +/- 2106); (1474 +/- 816) vs (3355 +/- 2042) respectively, (all P < 0.01). Western blot analysis demonstrated that PDCD5 expression was significantly decreased in the AML cells, as compared with normal cells. It is concluded that PDCD5 expression in MNC in untreated AML patients is lower than that in the normal. PDCD5 expression in the marrow granulocytes, monocytes, blast cells, and lymphocytes of untreated AML patients is significantly lower than that in the normal. It suggests that the abnormally low expression of PDCD5 may be involved in the pathogenesis of AML.

[Analysis of immunophenotype and leukemia associated immunophenotype in 610 patients with acute myeloid leukemia].

OBJECTIVE: To analyze the immunophenotype and leukemia associated immunophenotype (LAIP) of leukemia cells from patients with acute myeloid leukemia (AML) in minimal residual disease (MRD) detection. METHODS: Four-color multiparametric flow cytometry (FCM) with CD45/SSC gating was used to determine the expression of the following antibodies of CD7, CD117, CD33, CD34, CD10, CD19, CD56, CD38, CD13, CD14, CD64, CD9, CD16, CD2, CD5, CD11b, CD123, HLA-DR in 610 AML patients and 20 normal bone marrow (NBM) samples. RESULTS: The mean percentages of CD34+ and CD117+ side scatter low (SSC(low)) cells in NBM mononuclear cells (BMMNCs) were (0.35 +/- 0.15)% and (0.76 +/- 0.31)%, respectively. The majority (84% -95%) of CD34+ SSC(low) cells co-expressed CD13, CD33, CD38, CD117 and HLA-DR. 33% and 20% of CD34+ SSC(low) cells were CD15+ and CD9, respectively. Only a small proportion (< 10%) of CD34 SSC(low) cell co-expressed CD11b, CD56, CD19 and CD64, while co-expressed CD7 was 12%. In CD117 SSC(low) cells, the relative proportions of CD19+, CD11b+, CD56+ and CD7+ were less than 10%, while CD9+ was 14%. CD38+ and HLA-DR+ were 87% and 91%, respectively. The expressions of CD15, CD34, CD13 and CD33 on CD117 SSC(low) cells were between 30% and 50%. In AML patients, most cases were CD117+ (95.08%), CD38+ (94.74%), CD9+ (84.93%), CD33+ (84.60%), HLA-DR+ (77.23%) and CD13 (75.25%). The proportions of CD64+ and CD34+ were 64.41% and 59.51%, and that of CD15 and CD11b+ were 43.06% and 22.07%, respectively. 86.39% of AML patients were found to have at least one LAIP, the highest incidence being in AML-M1 and M3 subtypes and the lowest in AML-M4Eo subtype. The cross-lineage antigen and asynchronous antigen expression were the most frequent aberrant phenotypes. CD7, CD19 and CD56 expressing on CD34+ cells were major cross-lineage antigen. For asynchronous antigen expression, CD34+ CD64+, CD117+ CD11b+ , CD34+ CD38(-/dim) and CD34+ HLA-DR(-/dim) were seldom expressed on normal BMMNCs (about 0.01%), and the logarithm difference between AML and NBM was larger than 3.0, being the more sensitive LAIP. CONCLUSION: MRD detection by multiparameter flow cytometry can be applied to more than 80% of AML patients.

[Leukemia-associated immunophenotypes in 415 childhood and adult patients with B lineage acute lymphoblastic leukemia by multiparametric flow cytometry analysis].

To evaluate the significance of FCM in minimal residual disease (MRD) detection, the immunophenotyping and leukemia-associated immunophenotypes (LAIP) of leukemia cells from 273 adult and 142 childhood patients with B lineage acute lymphoblastic leukemia (B-ALL) were detected by four to six antibody combinations of 4-color CD45/SSC gating multiparametric flow cytometry (FCM). The results showed that the B-ALL patients could be classified into 4 subtypes based on different expression CD34 and CD10: subtype I (CD34(+)/CD10(-)), subtype II (CD34(+)/CD10(+)), subtype III (CD34(-)/CD10(+)), subtype IV (CD34(-)/CD10(-)). The LAIP was observed in 100% and 92% patients of subtype I and subtype II, respectively, whereas only 79.2% in subtype III. The incidence of LAIP in total B-ALL cases was 90% by using the antibodies detected in this investigation. There was no significantce different for incidence of LAIP between adult and pediatric patients. LAIP was observed in 77.6% of patients by labeling only CD34/CD10/CD19/CD45 4-color antibody combination. It is concluded that in 90% of childhood and adult B-ALL patients LAIP can be found, which suits MRD detection by multiparameter flow cytometry.

[Clinical significance for minimal residual disease detection by 4 color flow cytometry in adult and childhood B lineage acute lymphoblastic leukemia].

OBJECTIVE: To evaluate the clinical significance for minimal residual disease (MRD) detection by 4 color flow cytometry in B lineage acute lymphoblastic leukemia (B-ALL). METHODS: MRD was analyzed and followed up by using two panels of 4 color antibodies, mainly CD34/CD10/CD45/CD19, in 671 consecutive bone marrow specimens and 1 cerebrospinal fluid from 98 B-ALL patients. In 26 cases of them the immunophenotyping informations at diagnosis were not available. RESULTS: Of 671 bone marrow samples, 579 were MRD negative with leukemic cells below 0.0001 and 93 were MRD positive with leukemic cells over 0.0001. Of 93 MRD positive samples, leukemic cells below 0.05 were found in 64 bone marrow samples, meanwhile in the other 29 samples leukemic cells were over 0.05. Twenty patients relapsed, 19 were bone marrow relapse and one center nerves system. Fifteen of them were found MRD positive 7 - 17 weeks before relapse including 6 patients having no immunophenotyping data at diagnosis. The percentages of leukemia cells in these 15 patients were all over 0.0001. Two relapsed patients were MRD negative in 3 and 9 months before relapse, respectively. Two relapsed after MRD monitoring stopped. If MRD level was > 0.0001 at the end of induction chemotherapy and 12 weeks of treatment, the rate of relapse was 50% (6/12), while, it was 7.5% (3/40) in MRD negative patients (P = 0.000). CONCLUSION: Relapses can be predicted by MRD monitoring, if MRD was positive in the early phase of treatment, the risk of relapse was higher. Based on the characteristics of B cells ontogeny, MRD detection can be done independently of immunophenotypic information at diagnosis.

[Comparison of the immunophenotype of patients with B lineage acute lymphoblastic leukemia at diagnosis and relapse].

OBJECTIVE: To compare the leukemia-associated immunophenotypes (LAIP) in patients with B lineage acute lymphoblastic leukemia (B-ALL) at diagnosis and relapse, and investigate its implications for minimal residual disease (MRD) detection. METHODS: The immunophenotype of leukemia cells from 410 newly diagnosed and 6 relapsed patients with B-ALL were detected by four to six antibody combination, mainly CD34/CD10/CD45/CD19 of 4-color CD45/SSC gating flow cytometry (FCM). RESULTS: The proportion of CD45 under-expressed or negative in relapsed patients was much higher than that in newly diagnosed patients, being 69.2% and 37.8% respectively. Immunophenotypic changes occurred in 9 relapsed patients (including 8 hematological relapse and 1 central nerves system relapse) when analyzed by paired samples analysis at diagnosis vs at relapse: 4 cases showed CD45 down-modulation and 2 up-modulation; 4 CD34 down-modulation and 2 CD10 up-modulation, while the expression of CD19 remained no change. MRD was observed in all 7 cases of hematological relapse 2 - 4 months before relapse, and the immunophenotype of MRD cells was the same as that in relapse. CONCLUSION: A high frequency of immunophenotypic changes occurred at relapse and even in MRD before relapse, however the accuracy of MRD monitoring seemed not affected by the FCM strategy used in this investigation.

[Application of CD123 in detection of minimal residual disease in acute promyelocytic leukemia].

The study was aimed to investigate the role and significance of CD123 with other immunological markers in detecting minimal residual disease (MRD) of APL patients. The immunophenotypes of 186 newly diagnosed APL patients and the percentages of cells identical with APL cell immunophenotypes in 20 normal bone marrow samples were analyzed using four-color flow cytometry. MRD in 172 specimens were monitored by mainly using CD34/CD117/CD123/HLA-DR four-color antibody panels, meanwhile 18 specimens were analyzed with the second antibody combination: CD9/CD117/CD34/CD33, simultaneously and the results were compared with real-time PCR. One hundred and sixteen of 172 bone marrow (BM) or peripheral blood (PB) specimens were from follow-up 19 newly diagnosed APL patients and the rest 56 samples were from 47 patients treated 3 to 24 months later. Among them, 117 samples and 55 samples were collected after achieving morphologic complete remission (mCR) and before achieving mCR respectively. The results of immunophenotyping demonstrated that except CD9, CD33 and CD117 were high-expressed and CD34 and HLA-DR were rarely expressed, the CD123 was expressed in 30/30 (100%) APL patients. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) and CD117(+)CD34(-)CD9(+)CD33(+) cells in nucleated cells were 0.066% +/- 0.012% and 0.089% +/- 0.066% in 20 normal bone marrow samples. The median time of achieving morphology complete remission in 19 APL patients was 4 weeks (3 - 6 weeks). The median time of FCM and PCR results turned to be negative in 13 APL patients was 7.5 weeks (5 - 11) and the median time of PCR results turned to be negative in 11 APL patients was 8 weeks (5 - 12). 41/117 (35.04%) samples were MRD positive by FCM after achieving mCR. The ratio of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells was < 5% in 33 specimens, but > 5% in another 8 specimens, their median percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells were 0.48% (range 0.02% - 4.70%) and 9.02% (range 5.26% - 18.14%) respectively. The median relative percentages of CD123(+)HLA-DR(-) cells in CD117(+)CD34(-) population were 63.59% (range 15.11% - 98.36%) and 86.77% (range 63.29% - 92.62%) respectively. In FCM MRD positive samples, 95.9% (93/97) were PCR positive, the false positive rate of FCM and the false negative rate of PCR were 4.1% (4/97) and 8.75% (7/93) respectively. In FCM negative samples, 92% (69/75) were PCR negative and 8% (6/75) were PCR positive. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells in 116 consecutive specimens and 117 specimens of mCR were related to PML/RARalpha quantified by real-time PCR (r = 0.824, P < 0.001 and r = 0.754, P < 0.001 respectively). It is concluded that the detection of APL patients by means of two sets of antibody panels is simple and suitable, which is complementary to PCR in monitoring MRD of APL patients.