Background: Chronic fatigue syndrome (CFS) is an illness of unknown origin that may have familial risks. Low natural killer (NK) lymphocyte activity was proposed as a risk for familial CFS in 1998. Since then, there have been many studies of NK lymphocytes in CFS in general populations but few in familial CFS. Antibody-dependent cell-mediated cytotoxicity (ADCC) by NK lymphocytes helps control viral infections. ADCC is affected by variant CD16A receptors for antibody that are genetically encoded by FCGR3A. Methods: This report characterizes ADCC effector NK cell numbers, ADCC activities, and FCGR3A variants of five families each with 2-5 CFS patients, their family members without CFS and unrelated controls. The patients met the Fukuda diagnostic criteria. We determined: CD16Apositive blood NK cell counts; EC50s for NK cell recognition of antibody; ADCC lytic capacity; FCGR3A alleles encoding CD16A variants, ROC tests for biomarkers, and synergistic risks. Results: CFS patients and their family members had fewer CD16Apositive NK cells, required more antibody, and had ADCC that was lower than the unrelated controls. CFS family members were predominantly genetically CD16A F/F s for the variant with low affinity for antibodies. ROC tests indicated unsuitability of ADCC as a biomarker for CFS because of the low ADCC of family members without CFS. Familial synergistic risk vs. controls was evident for the combination of CD16Apositive NK cell counts with ADCC capacity. Conclusions: Low ADCC may be a risk factor for familial CFS. Furthermore, characterization of familial CFS represents an opportunity to identify pathogenic mechanisms of CFS.

NK cell ADCC supports monoclonal antibody anti-tumor therapies. We investigated serial ADCC and whether it could be predicted by NK phenotypes, including expression of CD16A, CD2 and perforin. CD16A, the NK receptor for antibodies, has AA158 valine or phenylalanine variants with different affinities for IgG. CD2, a costimulatory protein, associates with CD16A and can augment CD16A-signaling. Pore-forming perforin is essential for rapid NK-mediated killing. NK cells were monitored for their ADCC serial killing frequency (KF). KF is the average number of target cells killed per cell by a cytotoxic cell population. KF comparisons were made at 1:4 CD16pos NK effector:target ratios. ADCC was toward Daudi cells labeled with 51Cr and obinutuzumab anti-CD20 antibody. CD16A genotypes were determined by DNA sequencing. CD2, CD16A, and perforin expression was monitored by flow cytometry. Serial killing KFs varied two-fold among 24 donors and were independent of CD16A genotypes and perforin levels. However, high percentages of CD2pos of the CD16Apos NK cells and high levels of CD16A were associated with high KFs. ROC analysis indicated that the %CD2pos of CD16Apos NK cells can predict KFs. In conclusion, the extent of serial ADCC varies significantly among donors and appears predictable by the CD2posCD16Apos NK phenotype.

BACKGROUND:The search for a biomarker specific for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) has been long, arduous and, to date, unsuccessful. Researchers need to consider their expenditures on each new candidate biomarker. In a previous study of antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer lymphocytes, we found lower ADCC for ME/CFS patients vs. unrelated donors but ruled against low ADCC as a biomarker because of similar ADCC for patients vs. their family members without ME/CFS.OBJECTIVE:We applied inclusion of family members without ME/CFS, from families with multiple CFS patients, as a second non-ME/CFS control group in order to re-examine inflammation in ME/CFS.METHOD:Total and CD16A-positive 'non-classical' anti-inflammatory monocytes were monitored.RESULTS:Non-classical monocytes were elevated for patients vs. unrelated healthy donors but these differences were insignificant between patients vs. unaffected family members.CONCLUSIONS:Inclusion of family members ruled against biomarker considerations for the monocytes characterized. These pilot findings for the non-classical monocytes are novel in the field of ME/CFS. We recommend that occupational therapists advocate and explain to family members without ME/CFS the need for the family members' participation as a second set of controls in pilot studies to rapidly eliminate false biomarkers, optimize patient participation, and save researchers' labor.

ADCC (antibody-dependent cell-mediated cytotoxicity) is dependent on the varying capacity of NK cells to kill, the affinities of FCGR3A -encoded CD16A receptors for antibody, and the presence of antigen-specific antibodies. In vivo ADCC depends on the number of CD16A receptor-positive NK cells in blood. We hypothesized that low ADCC cell function or low effector cell numbers could be biomarkers or risk factors for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We measured NK cell ADCC lytic capacity and antibody recognition, CD16Apositive NK cells/µl blood, and FCGR3A homozygosity for the F allele that encodes low affinity CD16A antibody receptors. ME/CFS patients met the Fukuda 1994 diagnostic criteria. In this pilot report, we examined 5 families, each with 2 to 5 ME/CFS patients, and compared 11 patients, 22 family members without ME/CFS, and 16 unrelated healthy controls. ADCC was measured as CX1:1 cytotoxic capacity (the percentage of 51 Cr-Daudi tumors with obinutuzumab anti-CD20 antibody that were killed at a 1:1 ratio of CD16Apos NKs to Daudis) and CX-slope. Individual CX1:1 capacities varied from 16.2% to 81.8% and were comparable between patients and unaffected family members, while the ADCC of both family groups was lower than the unrelated healthy controls. The lack of difference between patients and their unaffected family members indicates that low ADCC is unsuitable as a diagnostic biomarker for ME/CFS. Familial CD16Apos NK blood cell counts were lower than unrelated healthy controls. The potential for synergistic effects of combined low CX1:1 and low effector cell counts occurring in the same individual was 24-fold greater for CFS family members than for unrelated controls. FCGR3A of the families was predominantly F/F homozygous, correlating with the observed low EC50 for NK recognition of target cell-bound antibody. In summary, low ADCC is unsuitable as a biomarker, but could be a familial risk factor, for ME/CFS.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by fatigue that does not improve with rest and worsens with exertion. ME/CFS may involve inflammation in the brain and/or be associated with viral infections. Increased numbers of pro-inflammatory monocytes in blood are a hallmark of infection. Biomarkers for, and information about, the CFS disease process are greatly needed. Here we queried circulating monocytes. In a pilot study, CFS patients and family members without CFS from three families were compared in order to reduce genetic and environmental variables. The patients conformed to the Fukuda 1994 standards. Unrelated healthy control subjects matched to the patients were included in the study. Blood monocytes were stained and analyzed by flow cytometry: counted with TruCountR beads, or stained after isolation and overnight culture of isolated peripheral blood monocytes. M1 pro-inflammatory monocytes are CD16A negative, while anti-inflammatory monocytes are CD16A positive; CD2 is a marker for dendritic monocytes, and changes in forward scatter could indicate in vitro activation. Notably, there were statistically significant differences between the lower percentages of M1 monocytes of the CFS patients compared to the controls; however, these differences were not significant between the patients and their family members without CFS. Also, the CD16A MFIs of the patients' monocytes were lower compared to controls but not to family members. One conclusion is that it is important to include family members without CFS as a control group in the search for biomarkers to diagnose CFS.

SUMMARY Physiological interconversion between specialized cell types has only been described in a few mammalian tissues and the mechanisms remain obscure. Using genetic lineage tracing during postnatal development and in-vitro models we demonstrate conversion of gastric interstitial cells of Cajal (ICC), regulatory cells that electrically pace phasic contractions and mediate nitrergic and cholinergic neural control of smooth muscle cells, into phenotypically distinct “fibroblast-like” interstitial cells (FLC), which only mediate purinergic signaling. Mechanistically, we find this transition to be epigenetically governed by H3K27 trimethylation of cell identity-related promoters whose susceptibility to repression is predicted by H3K27 acetylation patterns in ICC. The phenotypic switch was reversible by inhibition, knockdown or in-vivo genomic inactivation of the polycomb H3K27 methyl-transferase Ezh2. These results demonstrate a role for Ezh2-mediated epigenetic repression in physiological mammalian transdifferentiation and identify FLC as a reserve from which ICC can potentially be restored in common gastrointestinal disorders where ICC are depleted. GRAPHICAL ABSTRACT HIGHLIGHTS Gastric pacemaker cells (ICC) transdifferentiate into quiescent cells (FLC) in vivo ICC-to-FLC shift switches neural control from nitrergic/cholinergic to purinergic Ezh2-mediated H3K27me3 represses cell-identity genes during ICC-to-FLC transition Ezh2 inhibition restores ICC numbers, phenotype and function eTOC BLURB Syed et al. find aging to cause transdifferentiation of gastric pacemaker cells (interstitial cells of Cajal, ICC), which also communicate cholinergic and nitrergic neurotransmission to smooth muscle cells, into quiescent “fibroblast-like cells” (FLC), which only mediate purinergic signals. This switch is governed by Ezh2, whose inhibition can reverse ICC depletion.

Natural killer (NK) lymphocyte ADCC supports anti-viral protection and monoclonal antibody (mAb) anti-tumor therapies. To predict in vivo ADCC therapeutic responses of different individuals, measurement of both ADCC cellular lytic capacity and their NK cellular receptor recognition of antibodies on ‘target’ cells are needed, using clinically available amounts of blood. Twenty ml of blood provides sufficient peripheral blood mononuclear cells (PBMCs) for the new assay for lytic capacity described here and for an antibody EC50 assay for Fc-receptor recognition. For the lytic capacity assay, we employed flow cytometry to quantify the CD16A IgG Fc-receptor positive NK effector cells from PBMCs to avoid loss of NKs during isolation. Targets were 51Cr-labeled Daudi B cells pretreated with excess obinutuzumab type 2 anti-CD20 mAb and washed; remaining free mAb was insufficient to convert B cells in the PBMCs into ‘targets’. We calculated: the percentage Daudis killed at a 1:1 ratio of CD16A-positive NK cells to Daudis (CX1:1); lytic slopes; and ADCC50 lytic units. Among 27 donors, we detected wide ranges in CX1:1 (16–73% targets killed) and in lytic slopes. Slope variations prevented application of lytic units. We recommend CX1:1 to compare individuals' ADCC capacity. CX1:1 was similar for purified NK cells vs. PBMCs and independent of CD16A V & F genotypes and antibody EC50s. With high mAb bound onto targets and the high affinity of obinutuzumab Fc for CD16A, CX1:1 measurements discern ADCC lytic capacity rather than antibody recognition. This assay allows ADCC to be quantified without NK cell isolation and avoids distortion associated with lytic units.

Platelet-derived growth factor receptor alpha (PDGFRα)+ cells are distributed into distinct morphological groups within the serosal, muscular, and submucosal layers as well as the myenteric and deep muscular plexi. PDGFRα+ cells directly interact with interstitial cells of Cajal (ICC) and smooth muscle cells (SMC) in gastrointestinal smooth muscle tissue. These three cell types, SMC, ICC, and PDGFRα+ cells (SIP cells), form an electrical syncytium, which dynamically regulates gastrointestinal motility. We have previously reported the transcriptomes of SMC and ICC. To complete the SIP cell transcriptome project, we obtained transcriptome data from jejunal and colonic PDGFRα+ cells. The PDGFRα+ cell transcriptome data were added to the Smooth Muscle Genome Browser that we previously built for the genome-scale gene expression data of ICC and SMC. This browser provides a comprehensive reference for all transcripts expressed in SIP cells. By analyzing the transcriptomes, we have identified a unique set of PDGFRα+ cell signature genes, growth factors, transcription factors, epigenetic enzymes/regulators, receptors, protein kinases/phosphatases, and ion channels/transporters. We demonstrated that the low voltage-dependent T-type Ca2+ channel Cacna1g gene was particularly expressed in PDGFRα+ cells in the intestinal serosal layer in mice. Expression of this gene was significantly induced in the hyperplasic PDGFRα+ cells of obstructed small intestine in mice. This gene was also over-expressed in colorectal cancer, Crohn's disease, and diverticulitis in human patients. Taken together, our data suggest that Cacna1g exclusively expressed in serosal PDGFRα+ cells is a new pathological marker for gastrointestinal diseases.

Transcriptome-scale data can reveal essential clues into understanding the underlying molecular mechanisms behind specific cellular functions and biological processes. Transcriptomics is a continually growing field of research utilized in biomarker discovery. The transcriptomic profile of interstitial cells of Cajal (ICC), which serve as slow-wave electrical pacemakers for gastrointestinal (GI) smooth muscle, has yet to be uncovered. Using copGFP-labeled ICC mice and flow cytometry, we isolated ICC populations from the murine small intestine and colon and obtained their transcriptomes. In analyzing the transcriptome, we identified a unique set of ICC-restricted markers including transcription factors, epigenetic enzymes/regulators, growth factors, receptors, protein kinases/phosphatases, and ion channels/transporters. This analysis provides new and unique insights into the cellular and biological functions of ICC in GI physiology. Additionally, we constructed an interactive ICC genome browser (http://med.unr.edu/physio/transcriptome) based on the UCSC genome database. To our knowledge, this is the first online resource that provides a comprehensive library of all known genetic transcripts expressed in primary ICC. Our genome browser offers a new perspective into the alternative expression of genes in ICC and provides a valuable reference for future functional studies.

Antibody-dependent cell-mediated cytotoxicity (ADCC) supports anti-viral protection and monoclonal antibody anti-tumor therapies. We used two assays to compare ADCC of healthy donors: CX@1:1 (the percentage cells killed at a 1:1 ratio of CD16Apositive NK cells to ‘target’ cells that were pre-labeled with saturating antibody); and EC50 (the effective concentration of antibody that supports 50% of maximal ADCC). CX@1:1 measures lytic capacity while the EC50 measures cellular recognition; there was no correlation between the 2 assays though we observed 4.5-fold differences among donors in each assay. We correlated ADCC with 5 cytometric parameters (the median fluorescent intensities [MFIs] of the CD16A IgG Fc-receptor; the %NK cells positive & MFIs of the adhesion molecule CD2; the MFIs of perforin; and the %CD16Apositive of CD56positive NK cells) and with CD16A V&F genotypes that affect FcR affinity. For CX@1:1, the best donor killed 73% of targets while the worst killed 16% and perforin levels correlated weakly (P=0.11). Unexpectedly and inexplicably, the %CD16Apositive cells among NK cells correlated (P<0.05). CD16A and CD2 were dissociated from CX@1:1 as might be expected for the high antibody concentrations. For EC50s, there were no statistical correlations except for the expected difference between V/V &V/F vs. F/F genotypes, though the P=0.11. We conclude that substantial individual differences in ADCC per CD16Apositive NK cell are determined by parameters other than cell surface receptors and perforin. The unanticipated correlation of %CD16Apositive NK cells with CX@1:1 has potential as a surrogate marker for ADCC function.

This paper presents a continuous-flow cell screening device to isolate and separate microalgae cells (Chlamydomonas reinhardtii) based on lipid content using high frequency (50 MHz) dielectrophoresis. This device enables screening of microalgae due to the balance between lateral DEP forces relative to hydrodynamic forces. Positive DEP force along with amplitude-modulated electric field exerted on the cells flowing over the planar interdigitated electrodes, manipulated low-lipid cell trajectories in a zigzag pattern. Theoretical modelling confirmed cell trajectories during sorting. Separation quantification and sensitivity analysis were conducted with time-course experiments and collected samples were analysed by flow cytometry. Experimental testing with nitrogen starved dw15-1 (high-lipid, HL) and pgd1 mutant (low-lipid, LL) strains were carried out at different time periods, and clear separation of the two populations was achieved. Experimental results demonstrated that three populations were produced during nitrogen starvation: HL, LL and low-chlorophyll (LC) populations. Presence of the LC population can affect the binary separation performance. The continuous-flow micro-separator can separate 74% of the HL and 75% of the LL out of the starting sample using a 50 MHz, 30 voltages peak-to-peak AC electric field at Day 6 of the nitrogen starvation. The separation occurred between LL (low-lipid: 86.1% at Outlet # 1) and LC (88.8% at Outlet # 2) at Day 9 of the nitrogen starvation. This device can be used for onsite monitoring; therefore, it has the potential to reduce biofuel production costs.

We present a method to quantify and enhance separation of binary cells mixture in the microfluidic device using high frequency dielectrophoresis (>20 MHz). At these frequencies, the DEP response depends primarily on the dielectric properties of the cytoplasm. In order to achieve efficient separation, there must be a difference in the intrinsic dielectric properties of the populations to be sorted. For algae cells, the shift in high frequency dielectrophoresis response during lipid accumulation can be used as a basis of separation. We defined a separability parameter based on the expected difference in the dielectrophoresis responses of the algae cells. Chlamydomonas reinhardtii cells were cultured in regular media and then the same cells were cultured under nitrogenfree conditions to accumulate neutral (non-polar) lipids. Separability of microalgae cells with different lipid content via high frequency dielectrophoresis were investigated by a thin needle shaped electrodes patterned by standard photolithographic and wet etching procedures. Experimental separability factors were measured by estimation of relative lipid content with BODIPY 505/515 fluorescence dye and calculating the area-weighted intensity average of fluorescent images. Theoretical separability parameter was calculated using analytical analysis of single shell model by MATLAB. Theoretical and experimental separability parameters, as tools to determine the optimal separation method, were calculated for microalgae cells with different lipid content. This objective function was maximized in the range of 35–45 MHz for C. reinhardtii cells after 21 days of lipid accumulation in a static separation. In order to design a continuous cell sorter device, the theoretical separation factor was maximized based on differences in the magnitude or the direction of the DEP force.

trascript annotation of all gastrointestinal samples

Genome-scale expression data on the absolute numbers of gene isoforms offers essential clues in cellular functions and biological processes. Smooth muscle cells (SMCs) perform a unique contractile function through expression of specific genes controlled by serum response factor (SRF), a transcription factor that binds to DNA sites known as the CArG boxes. To identify SRF-regulated genes specifically expressed in SMCs, we isolated SMC populations from mouse small intestine and colon, obtained their transcriptomes, and constructed an interactive SMC genome and CArGome browser. To our knowledge, this is the first online resource that provides a comprehensive library of all genetic transcripts expressed in primary SMCs. The browser also serves as the first genome-wide map of SRF binding sites. The browser analysis revealed novel SMC-specific transcriptional variants and SRF target genes, which provided new and unique insights into the cellular and biological functions of the cells in gastrointestinal (GI) physiology. The SRF target genes in SMCs, which were discovered in silico, were confirmed by proteomic analysis of SMC-specific Srf knockout mice. Our genome browser offers a new perspective into the alternative expression of genes in the context of SRF binding sites in SMCs and provides a valuable reference for future functional studies.

Microparticulate β-glucan (MG) conjugated to vaccine antigen has been shown to serve as an effective adjuvant in vivo. To further study antigen presentation by MG:vaccine conjugates, bone marrow-derived dendritic cells (BMDC) were treated with MG conjugated to ovalbumin (OVA), then interacted with splenocytes from DO11.10 transgenic mice expressing an OVA peptide-specific T cell receptor. BMDC treated with MG:OVA induced significantly higher numbers of activated (CD25+CD69+) OVA-specific CD4+ T cells than BMDC treated with OVA alone. BMDC treated with MG:OVA upregulated CD86 and CD40 expression as well as MG alone, indicating that conjugation of OVA does not alter the immunostimulatory capacity of MG. Activation of CD8+ OVA-specific OT-1 cells showed that MG:OVA is also capable of enhancing cross-presentation by BMDC to CD8+ cytotoxic T cells. These results show that MG acts as an adjuvant to enhance antigen presentation by dendritic cells to naïve, antigen-specific CD4 and CD8 T cells.

Abstract Natural killer (NK) cytotoxicity of chronic fatigue syndrome (CFS) patients is decreased, as reported by several laboratories. This observation led us to query whether the NK cells also lack antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells have the IgG Fc-receptor CD16A that mediates ADCC. To assess potential ADCC effectors, we examined the peripheral blood of 11 CFS patients from a Lake Tahoe cohort that met the Fukuda standards for CFS and had low SF36 scores. Healthy controls were age- and gender-matched. We stained peripheral blood cells for CD16A with mAb clone 3G8 and for CD3, CD56, and perforin, then assessed the cells by flow cytometry. We found that the percentages of NK cells expressing CD16A were slightly lower for CFS patients 86.0+/-11.5% vs. controls 93.0+/-6.6% (P=0.08). The median fluorescent indices (MFIs) of CD16A were lower,72% of the values for the CFS patients, 9342+/-3233 vs. control 12929+/-4425, though not statistically significant (P<0.17). Intracellular staining for perforin in the CD16Apos cells was similar for patients and controls. Our data indicate that a larger sampling of CFS patients vs. controls is required to determine if the fractions of CD16Apos NK cells and the levels of CD16A on these cells differs. These two observations are both in the direction of compromising ADCC activity of patients. In addition, a bias in F over V allelic variants of CD16A might add to impairment, since the F allele confers reduced ADCC.