Absolute Cell Numbers Research Articles

Effects of Ketogenic Diet on Some Immunological Parameters.

&lt;b&gt;Background and Objective:&lt;/b&gt; A well-established treatment for childhood epilepsy, the Ketogenic Diet (KD) is increasingly gaining acceptance as a preventative tool for obesity. The current study aims to compare the effects of a ketogenic diet on some of the immunological parameters among obese individuals and contrast them with non-obese individuals. This includes finding the absolute numbers of white blood cells and estimating the efficacy of phagocytic cells and serum IgA concentration. &lt;b&gt;Materials and Methods:&lt;/b&gt; Twenty healthy persons were chosen for the study, with an age range between 18-45 years, a weight range before the ketogenic diet of 80-115 kg and after the ketogenic diet 60-80 kg with a height range between 155-180 cm during a period between 8-16 weeks. Their daily food was composed of high fats (80%), moderate protein (15%) and small amounts of carbohydrates (<5%). The control sample included twenty healthy subjects, their food consisted of different types of food carbohydrates (75-80%), proteins (10%) and fats (<10%). &lt;b&gt;Results:&lt;/b&gt; The results showed a significant increase in the White Blood Cells (WBC) count in KD individuals. Additionally, no significant difference was noticed in the absolute number of neutrophils and lymphocytes. Conversely, a significant increase in the absolute number of monocytes was observed. Moreover, the study showed a significant decrease in IgA concentration in KD individuals compared with control. &lt;b&gt;Conclusion:&lt;/b&gt; In the ketogenic system users indicated a rise in the number of monocytes and a decrease in the concentration of IgA. This provides a strong sign that autoimmune disorders are emerging.

The Influence of Low- and High-Negative-Pressure Liposuction and Different Harvesting Sites on the Viability and Yield of Adipocytes and Other Nucleated Cells

The volume effect of fat grafting is highly dependent on the presence of viable adipocytes and other nucleated cells within the lipoaspirate. We suspected that one of the crucial factors influencing cell viability is the negative pressure applied during the fat graft harvesting and the suitability of various harvest sites when compared to others. Despite much discussion, there is no consensus on the optimal negative pressure or the best site for harvesting so we designed an experiment to test this. Fat graft taken under low negative pressure (- 200 mmHg) or high negative pressure (- 700 mmHg) from the thigh or abdominal regions from 21 healthy human donors was evaluated. The principal variables studied were: a) total number and viability of nucleated cells, b) liposuction duration and c) blood admixture. Other variables studied were body mass index, the impact of age and enzymatic digestion. The absolute number and viability of nucleated cells and the blood admixture did not differ significantly between lipoaspirates obtained under different vacuum conditions or from different regions. The time taken to acquire the same volume of lipoaspirate was significantly increased using low negative pressure. The time taken to collect cells in the thigh region significantly increased with increasing BMI but this correlation was not found when harvesting in the abdominal region. The BMI and age did not impact the results in any of the measured variables. The enzymatic digestion rate was independent of the negative pressure used to harvest. Our results indicate that neither the negative pressure used nor the area chosen has any significant influence on the viability and yield of harvested cells. The time taken to obtain lipoaspirate using low pressure is significantly longer than when using high pressure. No significant difference was found in the value of blood admixture using different vacuum pressures, and no correlation exists between the body mass index and the cell viability or age of the patients and the time of liposuction. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine Ratings, please refer to Table of Contents or online Instructions to Authors www.springer.com/00266 .

FC 129CHANGES IN PERIPHERAL NK CELLS IN KIDNEY TRANSPLANT RECIPIENTS WITH AND WITHOUT HLA DSA

Abstract Background and Aims Antibody-mediated rejection (ABMR) is a frequent cause of renal allograft loss. There is increasing evidence of the role of Natural Killer (NK) cells in the establishment of ABMR damage. Our group described that patients with donor-specific antibodies (DSA) and ABMR present higher proportions of NKG2A+ NK cell subset in peripheral blood than those without HLA DSA or HLA antibodies. Method We selected 177 kidney transplant recipients (KT) with renal biopsies 2011-2017: 77 with ABMR (DSA+: 53, DSA-: 24) and 100 without ABMR (DSA+: 15, DSA-: 85). We assessed graft survival with a median time of follow-up since the renal biopsy of 53 months. In 138 KT we evaluated the peripheral blood NK cell immunophenotyping and its value as a prognostic biomarker. Results Graft survival was worse in ABMR-KT at the end of follow-up (p&amp;lt;0.001) independently of DSA detection (p=0.63). Regarding NK cell immunophenotyping, we observed a lower proportion and absolute NK cell count in ABMR+DSA+-KT and ABMR+DSA--KT compared with ABMR-DSA--KT (p=0.027, p=0.017). ABMR+DSA+-KT showed higher proportion of NKG2A+ NK cells compared with ABMR-DSA--KT (p=0.007). All ABMR+ patients, independently of DSA detection, presented lower absolute NKG2A- NK cell count in comparison with ABMR-DSA--KT (p=0.001, p=0.017). Finally, a proportion of NKG2A- &amp;lt;30% was associated with lower graft survival 36 months after graft biopsy with ABMR (p=0.067) (Figure). Conclusion Graft survival is worse in ABMR+ compared with ABMR- KT independently of DSA detection. Kidney transplant recipients with ABMR show reduced peripheral absolute numbers of NK cells and NKG2A- NK cells regardless of undetectable DSA. This NK cell phenotype associated with a worse medium-term graft survival in cases with ABMR.

Effects of endocrine-disrupting chemicals on hypothalamic oxytocin and vasopressin systems.

Exposures to endocrine disrupting chemicals (EDCs) perturb hormonal systems. EDCs are particularly problematic when exposure happens in the fetus and infant due to the high sensitivity of developing organisms to hormone actions. Previous work has shown that prenatal polychlorinated biphenyl (PCB) exposure disrupts hypothalamic development, reproductive physiology, mate preference behavior, and social behaviors in a sexually dimorphic manner. Based on evidence that EDCs perturb social behaviors in rodents, we examined effects of PCBs on the neuropeptides oxytocin (OXT) and vasopressin (AVP) that are involved in regulating these behaviors. Rats were exposed prenatally (gestational days 16 and 18) to the weakly estrogenic PCB mixture Aroclor 1221 (0.5 or 1 mg/kg), to estradiol benzoate (EB, a positive control), or to the vehicle (3% dimethyl sulfoxide). In adult (~P90) brains, we counted immunolabeled oxytocin and vasopressin cell numbers in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. EDCs did not change absolute numbers of oxytocin or vasopressin cells in either region, although there were some modest shifts in the rostral-caudal distribution. Second, expression of genes for these nonapeptides (Oxt, Avp), their receptors (Oxtr, Avpr1a), and the estrogen receptor beta (Esr2), was determined by qPCR. In the PVN, there were dose-dependent effects of PCBs in males (Oxt, Oxtr), and effects of EB in females (Avp, Esr2). In the SON, Oxt, and Esr2 were affected by treatments in males. These changes to protein and gene expression caused by prenatal treatments suggest that transcriptional and posttranscriptional mechanisms play roles in mediating how EDCs reprogram hypothalamic development.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

SummaryAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

POS1006 ABERRANT Th17 CELLS EXPANSION AND RISK FACTORS IN ANKYLOSING SPONDYLITIS PATIENTS COMPLICATED WITH CARDIOVASCULAR EVENTS

Background:The incidence of Ankylosing Spondylitis (AS) complicated with cardiovascular diseases (CVD) has increased in recent years [1]. However, identification of risk factors indicating the development of CAD in AS patients is lacking. Th17 cells are increasingly recognized to be important in atherogenesis [2]. However, the role of these cells in the pathogenesis of ankylosing spondylitis patients complicated with cardiovascular events remains elusive.Objectives:This study aimed to assess the level of circulating Th17 cells as well as other lymphocyte subsets such as Treg, Th, Ts, and NK cells in AS combined with CVD, and further to evaluate whether elevations in special PBMC subpopulations in AS patients indicate an increased risk of CVD.Methods:Samples were assessed from 141 AS patients hospitalized at the Second Hospital of Shanxi Medical University (60 AS patients combined with CVD and 81 AS patients without CVD) and 100 healthy controls. The absolute numbers of lymphocytes and CD4+ T cells in peripheral blood were determined using Flow Cytometer. The association between PBMC subpopulations and CVD development in AS patients were analyzed using multivariable logistic regression.Results:1. Compared with AS group, AS with CVD group exhibited significant increases in the number of Th17 cells (P=0.001) and Treg cells (P=0.046). The ratio of Th17/Treg was also increased (P=0.085).2. Analogous increases in the absolute number (P&lt;0.001) and frequency (P&lt;0.001) of Th1 cells, as well as the ratio of Th1/Th2 (P&lt;0.001) and Th1/Treg (P=0.004) were also present in AS with CVD patients, compared to those without CVD.3. Compared to HCs, 141 AS patients showed significantly decreased Treg cells (P&lt;0.012) and increased Th17/Treg (P=0.001).4. Logistic regression showed age (odds ratio: 1.09; 95% CI: 1.035-1.137), hypertension (odds ratio: 3.31; 95% CI: 1.152-9.528), diabetes (odds ratio: 8.03; 95% CI: 1.251-51.503), and elevated level of Th1 number (odds ratio: 1.01; 95% CI: 1.003-1.016) and DD (D-dimer) (odds ratio: 1.00; 95% CI: 1.000-1.002) were significantly correlated with the onset of CVD in AS patients.5. Smoke, increased Th17 level, and use of NSAIDS were also positively correlated with the onset of CVD although the P-values did not reach significant.Conclusion:Our data indicates aberrant expansion of Th17 cells in AS with CVD patients. Moreover, age, hypertension, diabetes, and increased level of Th1 in PBMC and DD are single independent risk factors for the presence of CVD in AS. The mechanisms of atherogenesis in AS may associate with the elevations in Th1 and Th17 cells. Imbalance of Th1/Th2 and Th17/Treg may be shared etiologic pathways of AS and CVD, providing attractive targets for the prevention and therapy of CVD development in AS patients.

POS0396 THE LEVEL OF PERIPHERAL REGULATORY T CELLS IS ASSOCIATED WITH THE CHANGES OF INTESTINAL MICROBIOTA IN PATIENTS WITH RHEUMATOID ARTHRITIS

Background:Rheumatoid arthritis (RA) is a systemic autoimmunity inflammation disease characterized with chronic aggressive arthritis and the presence of abnormal antibodies. Several observations showed that the breakdown of immune tolerance caused by many complex interactions was involved in the development of RA[1]. However, the pathogenesis of RA remained unclear. It has been confirmed that the imbalance of Th17 and Treg cells play a crucial role in destroying immune tolerance [2]. Besides, researches showed that intestinal microbiota can influence host immunity by acting on the immune cells to play pro-inflammatory or anti-inflammatory effect, and in turn immune system can also regulate the microbiota[3, 4]. Thus, a frontier point of view in the field of rheumatism, immune microecology, was proposed, which is a novel concept for the breakdown of immune tolerance. Studies have confirmed that there was an imbalance of intestinal microbiota in patients with RA [4]. But the relationship between the CD4+T subsets cells and intestinal microbiota in RA is unknown.Objectives:We detected and compared the absolute number of CD4+T cells subsets in the peripheral blood and the proportion or abundance of intestinal microbiota in patients with RA and healthy adults, and then analyzed the relationship between them to explore the role of CD4+T cells subsets and intestinal microbiota in the pathogenesis of RA.Methods:We collected the sample of stool and blood from 15 patients with RA hospitalized at the Second Hospital of Shanxi Medical University and 8 age and gender-matched healthy controls(HC). The absolute number of CD4+T cells subsets including Th1, Th2, Th17 and Treg cells were detected by flow cytometry. The 16S rRNA in the stool specimens were sequenced by the Roche/45 high-throughput sequencing platform. We analyzed whether there was correlarion between CD4+T subsets cells and intestinal microbiota.Results:Patients with RA had a higher level of Christensenellaceae and a lower level of Pseudomonadaceae as compared with those of HCs at the family level (p&lt;0.05). And at the genus level, the patients with RA had higher levels of Ruminococcus torques, Christensenellaceae R-7, Ruminiclostridium 9 and Ruminococcus 1 compared with those of HCs (p&lt;0.05) (Figure 1).And the Ruminococcus torques at the genus level was negative correlated with the absolute number of Treg cells (p&lt;0.001) (Figure 2).Conclusion:The results here suggested that there were different proportion or abundance of intestinal microbiota between the patients with RA andHCs. And the changes of intestinal microbiota such as Ruminococcus torques were associated with Treg cells, further indicating that the imbalance of intestinal microbiota in RA can destory the immune tolerance. The above results uncovered that the intestinal microbiota had immunomodulatory function, which may be the upstream mechanism participated in the pathogenesis of RA.

Peptidylprolyl isomerase C (Ppic) regulates invariant Natural Killer T cell (iNKT) differentiation in mice.

Peptidyl‐prolyl cis‐trans isomerase C (Ppic) is expressed in several bone marrow (BM) hematopoietic progenitors and in T‐cell precursors. Since the expression profile of Ppic in the hematoimmune system was suggestive that it could play a role in hematopoiesis and/or T lymphocyte differentiation, we sought to test that hypothesis in vivo. Specifically, we generated a Ppic‐deficient mouse model by targeting the endogenous locus by CRISPR/Cas9 and tested the requirement of Ppic in hematopoiesis. Several immune cell lineages covering BM progenitors, lymphocyte precursors, as well as mature cells at the periphery were analyzed. While most lineages were unaffected, invariant NKT (iNKT) cells were reduced in percentage and absolute cell numbers in the Ppic‐deficient thymus. This affected the most mature stages in the thymus, S2 and S3, and the phenotype was maintained at the periphery. Additionally, immature transitional T1 and T2 B lymphocytes were increased in the Ppic‐deficient spleen, but the phenotype was lost in mature B lymphocytes. In sum, our data show that Ppic is dispensable for myeloid cells, platelets, erythrocytes, αβ, and γδ T lymphocytes in vivo in the steady state, while being involved in B‐ and iNKT cell differentiation.

CX3CL1 Recruits NK Cells Into the Central Nervous System and Aggravates Brain Injury of Mice Caused by Angiostrongylus cantonensis Infection.

Background Angiostrongylus cantonensis (A. cantonensis), is a food-borne zoonotic parasite that can cause central nervous system (CNS) injury characterized by eosinophilic meningitis. However, the pathogenesis of angiostrongylosis remains elusive. Natural killer cells (NK cells) are unique innate lymphocytes important in early defense against pathogens. The aim of this study was to investigate the role of NK cells in A. cantonensis infection and to elucidate the key factors that recruit NK cells into the CNS.MethodsMouse model of A. cantonensis infection was established by intragastric administration of third-stage larvae. The expression of cytokines and chemokines at gene and protein levels was analyzed by qRT-PCR and ELISA. Distribution of NK cells was observed by immunohistochemistry and flow cytometry. NK cell-mediated cytotoxicity against YAC-1 cells was detected by LDH release assay. The ability of NK cells to secrete cytokines was determined by intracellular flow cytometry and ELISA. Depletion and adoptive transfer of NK cells in vivo was induced by tail vein injection of anti-asialo GM1 rabbit serum and purified splenic NK cells, respectively. CX3CL1 neutralization experiment was performed by intraperitoneal injection of anti-CX3CL1 rat IgG.ResultsThe infiltration of NK cells in the CNS of A. cantonensis-infected mice was observed from 14 dpi and reached the peak on 18 and 22 dpi. Compared with uninfected splenic NK cells, the CNS-infiltrated NK cells of infected mice showed enhanced cytotoxicity and increased IFN-γ and TNF-α production ability. Depletion of NK cells alleviated brain injury, whereas adoptive transfer of NK cells exacerbated brain damage in A. cantonensis-infected mice. The expression of CX3CL1 in the brain tissue and its receptor CX3CR1 on the CNS-infiltrated NK cells were both elevated after A. cantonensis infection. CX3CL1 neutralization reduced the percentage and absolute number of the CNS-infiltrated NK cells and relieved brain damage caused by A. cantonensis infection.ConclusionsOur results demonstrate that the up-regulated CX3CL1 in the brain tissue recruits NK cells into the CNS and aggravates brain damage caused by A. cantonensis infection. The findings improve the understanding of the pathogenesis of angiostrongyliasis and expand the therapeutic intervention in CNS disease.

Absolute Circulating Leukemic Cells as a Risk Factor for Early Bleeding Events in Patients with Non-High-Risk Acute Promyelocytic Leukemia.

BackgroundHemorrhagic complications are the most common cause of early death in patients with APL and remain a major challenge in the management of APL. Early fatal bleeding events occur not only in high-risk but also in non-high-risk acute promyelocytic leukemia (APL) patients with normal or low WBC counts.Objectives and MethodsTo demonstrate the role of the absolute number of circulating leukemic cells in early bleeding events in APL patients. Clinical and laboratory characteristics of 149 patients newly diagnosed with APL were obtained from medical records and retrospectively investigated.ResultsIn this study, circulating absolute leukemic cells were positively correlated with the WBC count (r=0.9813, p<0.001) in all patients with APL, and importantly, they were strongly associated with significant bleeding events in non-high-risk patients. Multivariate logistic regression analysis showed that the absolute number of leukemia cells was an independent risk factor for significant bleeding events in APL patients. A cut-off value of 2.59×109/L for circulating leukemic cells to predict significant bleeding events in APL patients was obtained by ROC curve analysis. We further confirmed that the significant bleeding rate of patients with non-high-risk APL was statistically increased when the absolute number of circulating leukemic cells was ≥2.59×109/L.ConclusionCirculating leukemic cell content has great clinical value for predicting early bleeding events in APL patients, especially in non-high-risk APL.

Vascular Endothelium‐Derived ET‐1 Regulates The Renal T Cell‐Macrophage Interplay Under High Salt Diet

The vasoactive peptide endothelin-1 (ET-1) is a key contributor to high salt-induced elevations of blood pressure and end-organ damage. In addition, ET-1 is known to be pro-inflammatory in the high salt setting. Although ET-1 was first described as produced by the vascular endothelium, we now know that there are other cellular sources, like kidney tubular cells or immune cells. However, the exact role of endothelium-derived ET-1 in regulating immune cell populations during high salt consumption remains unclear. We hypothesized that endothelium-derived ET-1 is critical in regulating the kidney inflammation that is associated with high salt consumption. Male vascular endothelial cell ET-1 knockout (VEET KO) and control floxed ET-1 mice were placed on 4.0% NaCl diet (HSD) for 3 weeks. Urine was collected for assessment of kidney injury, and the immunological status was evaluated in kidneys and spleen by flow cytometry analysis of live, singlet cells. After 3 weeks of HSD, VEET KO mice presented reduced urinary protein excretion compared to floxed ET-1 mice (floxed ET-1 vs. VEET KO: 8.59 ± 1.90 vs. 3.56 ± 0.87, pg/day; p=0.03; n=6/group), suggesting that vascular ET-1 is critical for developing high salt-induced kidney injury. Lack of endothelium-derived ET-1 resulted in a 53.9% reduction in the frequency of kidney TH17 cells (IL-17A+ CD4+ T cells; floxed ET-1 vs.VEET KO: 1.650.17 vs. 0.770.10, % IL-17A of CD4+; p=0.001; n=5-6/group) and a 77.9% reduction in kidney absolute TH17 cell count (3.10.5 vs. 0.850.22, x102 cells; p=0.008). Interestingly, the intra-renal frequency of T regulatory (Treg) cells was similar between the genotypes (floxed ET-1 vs. VEET KO: 12.81.58 vs. 12.31.29, %; p=0.78), but the absolute Treg cell number in the kidney was reduced in VEET KO mice (2.260.25 vs. 1.210.30, x103 cells, p=0.02); these findings suggest a potential role for vascular ET-1 in the regulation of HSD-induced Treg expansion/proliferation in this organ. In addition, VEET KO mice also presented significantly blunted frequency and absolute numbers of the innate-like IL-17A+ +TCR+ T cell population in the kidney (floxed ET-1 vs. VEET KO; % IL-17A of IL-17A+ + TCR+: 36.61.92 vs. 21.22.63, p=0.0009; absolute numbers: 2.510.41 vs. 1.020.39, x102, p=0.03). Examination of the kidney myeloid cell populations under HSD also revealed a reduced frequency of activated resident macrophages (F4/80hi CD64+: 25.14.52 vs. 10.51.44, %, p=0.01; F4/80hi CD86+: 85.32.38 vs. 78.131.69 %, p=0.03), while no differences were found in the activation of infiltrating macrophages (F4/80lo CD64+ and F4/80lo CD86+) in the kidney. Lymphoid and myeloid immune cell populations in the spleen were similar between genotypes. These data indicate a critical role for intra-renal endothelium-derived ET-1 in the cellular response of immune cell populations under HSD. More specifically, our data demonstrate that endothelium-derived ET-1 regulates the immune landscape during HSD consumption by activating pro-inflammatory TH17 and IL-17A+ +TCR+ T cells as well as resident macrophages specifically in the kidney.

Clinical and diagnostic value of the combination of lymphocyte count and creatine kinase in the detection of coronavirus 2019.

The present study aimed to investigate the diagnostic efficiency of the absolute number of lymphocytes (LYM) and creatine kinase (CK) levels in the diagnosis of coronavirus disease 2019 (COVID-19). For this, the clinical data from 84 patients with COVID-19 admitted to Tianjin Haihe Hospital (Tianjin, China) between January and February 2020 were collected. The patients were divided into the following groups: The common COVID-19 group (n=61) and severe COVID-19 group (n=23). In addition, 30 healthy subjects were included as a control group. The results demonstrated that the percentage of neutrophils (NEU%) was significantly increased, while the absolute number of white blood cells, LYM and the percentage of lymphocytes (LYM%) were significantly decreased in patients with COVID-19. Furthermore, in the severe group, the absolute number of red blood cells in female patients, the NEU%, the neutrophil-to-lymphocyte ratio (NLR) and the serum levels of interleukin-6 and C-reactive protein (CRP) were markedly elevated, while those of LYM and LYM% were significantly decreased (all P<0.05). In addition, in the receiver operating characteristics curve analysis for the combination of LYM + CK, the area under the curve values were 0.96 and 1.00, with a sensitivity of 95.08 and 100%, specificity of 86.67 and 100% and cut-off values of 0.42 and 0.50 for the common and severe COVID-19 group, respectively. The results indicated that the diagnostic efficiency of LYM + CK was higher than that of each single factor. Finally, a moderate correlation of lactate dehydrogenase with CRP and NLR (r=0.492 and 0.433, respectively; both P<0.05) was obtained. Overall, the results of the present study indicated that the values of LYM and CK were associated with the progression of COVID-19, suggesting that the combination of both factors may be of clinical diagnostic value for COVID-19.

High CD8/CD33 ratio in peritoneal metastatic lesions is associated with favorable prognosis in gastric cancer.

BackgroundTumor‐infiltrating lymphocytes (TILs) and other immune cells have been reported as a prognostic factor in several tumors, including gastric cancer, and they play an important role in antitumor effect at the primary site. There were few reports on the immune status in peritoneal metastatic lesions for gastric cancer.AimsThe aims of this study were to assess the prognostic significance of TILs (CD4, CD8, CD19, regulatory T cells [Tregs]), and myeloid‐derived suppressor cells (MDSCs) in peritoneal metastatic lesions.MethodsWe retrospectively investigated 60 patients for gastric cancer with peritoneal metastasis who were treated between 2009 and 2016 in our institute. Immunohistochemistry for CD4, CD8, CD19, FOXP3, and CD33 was performed in the peritoneal metastatic lesions. The absolute numbers of immune cells and ratios were evaluated, and the relationship between immune‐related marker and overall survival (OS) was investigated.ResultsA high infiltration of CD8+ lymphocytes or high CD8/CD33 ratio was a better prognosis for OS in univariate analysis using all immunologic variables (P = .012, P = .001). In multivariate analysis for clinical and immunologic variables, high CD8/CD33 ratio was identified as an independent prognostic factor for OS (Hazard ratio: 0.291, 95% confidence interval: 0.126‐0.670, P = .004).ConclusionHigh CD8/CD33 ratio and high infiltration of CD8+ lymphocytes in peritoneal metastatic lesions were favorable prognoses for gastric cancer patients with peritoneal metastasis. It is necessary to modify the immune microenvironment result to increase the level of CD8+ lymphocytes in the peritoneal metastatic lesions.

Ammonium Salts Promote Adaptive Changes of Rat Immune System to Ultimate Load in the Forced Swimming Model.

We studied the effect of different doses of ammonium chloride (ACl) and ammonium carbonate (ACr) on immunological parameters of the peripheral blood in rats during high-intensity exercise. Changes in the absolute and relative numbers of granulocytes, lymphocytes, natural killers, naive and mature effector cells one day after the end of the forced swimming cycle were found by using a hematological analyzer and a flow cytometer. Immunological indicators were analyzed relative to swimming duration on the last day of ultimate load. The revealed changes indicate the onset of the effector phase of the development of the inflammatory processes in the positive control group (physiological saline) and in rats receiving a higher dose of ACr (20 mg/kg), while administration of ACl prevented the development of inflammatory processes and shifts in the physiological balance of lymphocyte subpopulations. Immunological profiling indicates that ACl in a dose of 20 mg/kg most effectively improved physical performance in our forced swimming model.

Comparison of three qPCR-based commercial tests for detection of periodontal pathogens

In periodontal practice microbial results of periodontal test kits for identification of key pathogens are an aid in the treatment planning. Information on the performance of commercially available test kits is therefore essential for the clinician. In this retrospective analysis three commercially available qPCR kits for detection and quantification of selected periodontal bacterial species were compared, using 100 clinical samples from patients with untreated periodontitis. The analysis involved two separate comparisons in which kit A (LabOral Diagnostics, The Netherlands) was compared with kit B (Advanced Dental Diagnostics, The Netherlands), and with kit C (OralDent diagnostics, The Netherlands). Analytic procedures for detection and quantification of selected periodontal bacterial species were carried out according to the instructions of the laboratories. Kit A detected target species more often, and absolute numbers of bacterial cells were higher than with kit B. A high degree of similarity was found between the test outcomes by kit A and kit C. All three kits performed satisfactory but small and significant differences exist between kits.

Patterns of dendritic cell and monocyte subsets are associated with disease severity and mortality in liver cirrhosis patients

Liver cirrhosis is often complicated by an immunological imbalance known as cirrhosis-associated immune dysfunction. This study aimed to investigate disturbances in circulating monocytes and dendritic cells in patients with acute decompensation (AD) of cirrhosis. The sample included 39 adult cirrhotic patients hospitalized for AD, 29 patients with stable cirrhosis (SC), and 30 healthy controls (CTR). Flow cytometry was used to analyze monocyte and dendritic cell subsets in whole blood and quantify cytokines in plasma samples. Cirrhotic groups showed higher frequencies of intermediate monocytes (iMo) than CTR. AD patients had lower percentages of nonclassical monocytes than CTR and SC. Cirrhotic patients had a profound reduction in absolute and relative dendritic cell numbers compared with CTR and showed higher plasmacytoid/classical dendritic cell ratios. Increased plasma levels of IL-6, IL-10, and IL-17A, elevated percentages of CD62L+ monocytes, and reduced HLA-DR expression on classical monocytes (cMo) were also observed in cirrhotic patients. Patients with more advanced liver disease showed increased cMo and reduced tissue macrophages (TiMas) frequencies. It was found that cMo percentages greater than 90.0% within the monocyte compartment and iMo and TiMas percentages lower than 5.7% and 8.6%, respectively, were associated with increased 90-day mortality. Monocytes and dendritic cells are deeply altered in cirrhotic patients, and subset profiles differ between stable and advanced liver disease. High cMo and low TiMas frequencies may be useful biomarkers of disease severity and mortality in liver cirrhosis.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Although chimeric antigen receptor T (CAR-T) cell therapy achieves high remission rates, challenges (e.g., toxicity management and relapse prevention) remain. The major risks are cytokine release syndrome and related neurological toxicity. The influence of the CAR surface density on the efficacy/safety of CAR-T cell therapy and the factors determining CAR density were not elucidated comprehensively. Here, we discovered that the use of the MND promoter increased the transduction rate and reduced the CAR surface density. Additionally, MND-driven CAR-T cells had prolonged antileukemia activity in a mouse model. In an initial dual-armed anti-CD19 CAR-T cell pilot study (ClinicalTrials.gov: NCT03840317), eight and six subjects were infused with MND and EF1α promoter-driven autologous CAR-T cells (3 × 105 CAR-T cells/kg), respectively. MND subjects developed mild fever and lower interferon gamma (IFN-γ) concentrations than in the EF1A19 group. All but one subject in each cohort reached minimal residual disease (MRD)-negative complete remission after the first month of evaluation. These results represent the first comprehensive study on the promoter-driven modulation of CAR-T cell functionality. These findings encourage further evaluation of the potential of the MND promoter to drive CAR-T cells as a broadly applicable cellular product for anticancer immunotherapy.

Associations between peripheral blood lymphocyte subsets and clinical outcomes in patients with lung cancer treated with immune checkpoint inhibitor.

This study aimed to estimate peripheral blood lymphocyte subsets and programmed death receptor-1 positive (PD-1+) proportions of T cells, and their impact on progression free survival (PFS) and radiological response in lung cancer. From May 2018 to April 2020, 34patients of the Henan Tumor Hospital who were diagnosed with advanced lung cancer were recruited to this study. Peripheral blood lymphocyte subsets and PD-1+ proportions of T cells were assessed by flow cytometry before and after treatment with immune checkpoint inhibitors (ICIs). The associations among these parameters, and PFS and clinical response were estimated by survival analysis and Fishers' exact test, respectively. Several lymphocyte variables and biomarkers were found to be correlated with PFS and tumor response, as assessed using the Response Evaluation Criteria in Solid Tumors (RECIST). In all 34 lung cancer participants and a subgroup of 28 participants with non-small cell lung cancer (NSCLC), higher levels of natural killer (NK) cells and higher CD4+/CD8+ cell ratios before the ICIs treatment were associated with longer PFS. Moreover, CD4+ T cells were significantly correlated with radiological response in all 34 lung cancer participants. Of the 28 NSCLC participants, those with higher levels of CD4+ T cells, CD4+/CD8+ cell ratios, absolute numbers of NK cells, and lower levels of regulatory T cells (Tregs)before treatment had better tumor response. After 2 cycles of combined ICIs treatment, both the absolute numbers of CD4+ T cells and CD45+ lymphocytes were statistically associated with PFS after being adjusted for gender and neutrophil-lymphocyte ratio (NLR) [hazard ratio (HR) =0.23, P=0.015; HR=0.30, P=0.032, respectively]. The absolute numbers of CD45+, CD3+, and CD4+ T lymphocytes were associated with radiological response treated by ICIs (P=0.038). Our results suggested that the absolute number of NK cells and CD4+/CD8+ cells ratio before treatment could predict longer PFS and better radiological response in lung cancer patients treated with ICIs combination therapy. In addition, Tregs, as well as the other parameters in lymphocyte subsets, may also predict response.

Clinical and morphological differential diagnosis of congenital hyperinsulinism before the stage of immunohistochemical study of paraffin-embedded specimens of pancreas in children

Introduction.Differential diagnosis of various forms of congenital hyperinsulinism (CH) is required at the stage of urgent histological examination, but the difference between the exo- and endocrine parts of the pancreas in frozen sections is visualized very poorly.The objective was to search for differential diagnostic criteria of focal and diffuse forms of CH, suitable for urgent histological examination.Methods and materials. Pancreatic surgery material from 25 children with CH, of which 15 cases of focal form (FCH) and 10 cases of diffuse (DСH), and autopsy material from 10 children without CH (K). Frozen and paraffin sections stained with hematoxylin-eosin and histochemical stains were made from the tissue of the pancreas. The number of endocrinocytes with large nuclei was estimated in 10 fields of view for each case.Results. The average proportion of endocrinocytes with large nuclei was significantly (p &lt;0.01) higher compared to the control both with FCH (in the affected area) and DCH (FCH – (1.82±0.50) %, DCH – (4.68±1.66) %, K – (0.34±0.21) %). We found a tendency to detect a higher absolute number of cells with large nuclei in DCH ((4.95±1.24) DCH and (3.71±1.56) FCH). Staining with azure-eosin, picro Mallory and the PAS reaction gave unstable result, and staining with toluidine blue and May-Grunwald did not allow differentiating exo- and endocrine parts of the pancreas.Conclusion. An increase in the size of the endocrinocyte nuclei was the unsuitable criterion for the differential diagnosis of CH forms, and histochemical stains did not give a stable qualitative result. For urgent biopsy examination, new immunohistochemical methods are needed.

Impaired Cellular Immunity to SARS-CoV-2 in Severe COVID-19 Patients.

The high infection rate and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) make it a world-wide pandemic. Individuals infected by the virus exhibited different degrees of symptoms, and most convalescent individuals have been shown to develop both cellular and humoral immune responses. However, virus-specific adaptive immune responses in severe patients during acute phase have not been thoroughly studied. Here, we found that in a group of COVID-19 patients with acute respiratory distress syndrome (ARDS) during hospitalization, most of them mounted SARS-CoV-2-specific antibody responses, including neutralizing antibodies. However, compared to healthy controls, the percentages and absolute numbers of both NK cells and CD8+ T cells were significantly reduced, with decreased IFNγ expression in CD4+ T cells in peripheral blood from severe patients. Most notably, their peripheral blood lymphocytes failed in producing IFNγ against viral proteins. Thus, severe COVID-19 patients at acute infection stage developed SARS-CoV-2-specific antibody responses but were impaired in cellular immunity, which emphasizes on the role of cellular immunity in COVID-19.