Analysis of Immune Cell Subsets in Peripheral Blood from Patients with Engineered Stone Silica-Induced Lung Inflammation.

Silicosis caused by engineered stone (ES-silicosis) is an emerging worldwide issue characterized by inflammation and fibrosis in the lungs. To our knowledge, only a few reports have investigated leukocyte/lymphocyte subsets in ES-silicosis patients. The present study was designed to explore the proportions of the main lymphocyte subsets in ES-silicosis patients stratified into two groups, one with simple silicosis (SS) and the other with a more advanced state of the disease, defined as progressive massive fibrosis (PMF). The proportions of B (memory and plasmablasts) cells, T (helper, cytotoxic, regulatory) cells, and natural killer (NK) (regulatory and cytotoxic) cells were investigated by multiparameter flow cytometry in 91 ES-silicosis patients (53 SS patients and 38 PMF patients) and 22 healthy controls (HC). Although the total number of leukocytes did not differ between the groups studied, lymphopenia was observed in patients compared to healthy controls. Compared with those in healthy controls, the proportions of memory B cells, naïve helper T cells, and the CD4+/CD8+ T cells' ratio in the peripheral blood of patients with silicosis were significantly decreased, while the percentages of plasma cells, memory helper T cells, and regulatory T cells were significantly increased. For the NK cell subsets, no significant differences were found between the groups studied. These results revealed altered cellular immune processes in the peripheral blood of patients with ES-silicosis and provided further insight into silicosis pathogenesis.

Plasma Cytokine Profiling Reveals Differences between Silicotic Patients with Simple Silicosis and Those with Progressive Massive Fibrosis Caused by Engineered Stone.

Engineered stone silicosis has become an occupational epidemic disease that progresses rapidly to progressive massive fibrosis with respiratory failure and death, and there is no effective treatment. Silica deposition in the lung triggers a series of inflammatory reactions with the participation of multiple cytokines and cellular mediators whose role in the development and progression of the disease is largely unknown. We hypothesized that differences in plasma cytokine levels exist between patients diagnosed with simple silicosis (SS) and patients diagnosed with progressive massive fibrosis (PMF). Plasma samples from 91 ES silicosis patients, diagnosed and classified by chest radiography and/or high-resolution computed tomography with SS (n = 53) and PMF (n = 38), were assayed by multiplex assays for levels of 34 cytokines. Additionally, a healthy volunteer control group (n = 22) was included. Plasma levels of a high number of cytokines were significantly higher in subjects with silicosis than in healthy control subjects. Moreover, the levels of IL-1RA, IL-8, IL-10, IL-16, IL-18, TNF-α, MIP-1α, G-CSF and VEGF were significantly elevated in PMF compared to SS patients. This study shows that plasma cytokine levels differ between healthy people and silicosis patients, and some of them are also significantly elevated in patients with PMF compared with patients with SS, which could indicate their involvement in the severity of the disease, be considered as biomarkers and could be explored as future therapeutic targets for the disease.

Normal Cerebellar Growth by Using Three-dimensional US in the Preterm Infant from Birth to Term-corrected Age.

Purpose To establish cross-sectional and longitudinal reference values for cerebellar size in preterm infants with normal neuroimaging findings and normal 2-year neurodevelopmental outcome by using cranial ultrasonography (US). Materials and Methods This prospective study consecutively enrolled preterm infants admitted to a neonatal intensive care unit from June 2011 to June 2014 with a birth weight of less than or equal to 1500 g and/or gestational age (GA) of less than or equal to 32 weeks. They underwent weekly cranial US from birth to term-equivalent age and magnetic resonance (MR) imaging at term-equivalent age. The infants underwent neurodevelopmental assessments at age 2 years with Bayley Scales of Infant and Toddler Development, 3rd edition (BSID-III). Patients with adverse outcomes (death or abnormal neuroimaging findings and/or BSID-III score of <85) were excluded. The following measurements were performed: vermis height, craniocaudal diameter, superior width, inferior width, vermis area, and transcerebellar diameter. Statistical analyses were conducted by using multilevel analyses. Results A total of 137 infants with a mean GA at birth of 29.4 weeks (range, 25-32 weeks) were included. Transcerebellar diameter increased by 1.04 mm per week on average; vermis height and craniocaudal diameter increased by 0.55 mm and 0.59 mm, respectively. Superior vermian width increased by an average of 0.45 mm, whereas inferior vermian width increased by an average of 0.51 mm per week. Vermis area was found to increase by 0.22 cm2 per week on average. The sex effect was significant (female lower than male) for vermis height (P < .05), craniocaudal diameter (P < .05), inferior vermian width (P <. 05), and vermis area (P <. 05). Conclusion Cross-sectional and longitudinal reference values were established for cerebellar growth in preterm infants, which may be included in routine cranial US.

Ultrasound lineal measurements predict ventricular volume in posthaemorrhagic ventricular dilatation in preterm infants.

AIM: Posthaemorrhagic ventricular dilatation (PHVD) is monitored by conventional two-dimensional ultrasound (2DUS). The aims of this study were to determine the volume of the lateral ventricles using three-dimensional ultrasound (3DUS) in preterm infants with PHVD and to evaluate the relationship between volume and linear measurements. METHODS: Serial 2DUSs and 3DUSs were performed on preterm infants with PHVD admitted to the neonatal intensive care unit at Puerta del Mar Hospital, Cádiz, Spain, from January 2013 to December 2014. The ventricular index, anterior horn width and thalamo-occipital distance were used as ventricular lineal measurements. Ventricular volume was calculated offline. RESULTS: Serial ultrasounds from seven preterm infants were measured. Each linear measurement was significantly associated with volume, and an equation was obtained through a significant multilevel mixed-effects lineal regression model: ventricular volume (cm3 ) = -11.02 + 0.668*VI + 0.817*AHW + 0.256*TOD. Intra-observer and interobserver agreement was excellent with an intraclass correlation coefficient of 0.99. CONCLUSION: Lateral ventricular volumes of preterm infants with PHVD could be reliably determined using 3DUS. Ventricular volume could be accurately estimated using three lineal measurements. More studies are needed to address the importance of volume determination in PHVD.

Effect of Filters on the Noise Generated by Continuous Positive Airway Pressure Delivered via a Helmet.

INTRODUCTION: One of the problems that the delivery of continuous positive airway pressure (CPAP) via a helmet poses is the generation of noise. The objective of our study was to assess the effect that the use of filter has on sound pressure levels generated by the delivery of positive airway pressure at different gas flow rates. MATERIALS AND METHODS: Sound pressure levels generated by neonatal helmet CPAP delivery were measured at different gas flows (20, 30, and 40 l/min) with and without a breathing filter. Noise intensity was measured by installing microphones in the inner ear of dummy heads wearing helmets. RESULTS: The sound pressure level increased by 38% at a gas flow of 40 l/min, as compared to a gas flow of 20 l/min {74 dBA [interquartile range (IQR) 2,2] vs 52 dBA (IQR 5,9), respectively}. Using the breathing filter as a diffuser has a variety of effects on sound pressure levels according to the gas flow rate. CONCLUSION: The intensity of the noise generated by helmet delivery of positive airway pressure depends on the type of helmet used, gas flow, and use or not of a diffuser filter. Breathing filters with gas flows over 30 l/min might not be recommended since they would not attenuate but will rather amplify sound pressure.

Low-voltage pattern and absence of sleep-wake cycles are associated with severe hemorrhage and death in very preterm infants.

UNLABELLED: Amplitude integrated electroencephalogaphy (aEEG) is becoming an important tool for the assessment of cerebral activity in preterm newborns. Describing the relationship between early aEEG patterns and intraventricular hemorrhage (IVH) can improve our knowledge of neurological injury in the preterm newborn. The aim of this prospective study was to identify early changes in the aEEG in premature newborns that could be associated to severe neurological lesion/death. Preterm newborns with a birth weight ≤1,500 g and/or 32 weeks of gestation were included. aEEG monitoring was performed during the first 72 h of life. A qualitative analysis of the aEEG recordings was performed, based on continuity, sleep-wake cycles (SWCs), inferior lower margin amplitude (LMA), and bandwidth (BW). Key outcomes were severe IVH and/or death. Ninety-two subjects were included (mean gestational age 28 weeks). In 28.6 % of subjects with HIV III/IHP, a low-voltage pattern was observed. A statistically significant relationship was found between low-voltage tracings and death and neurological lesion/death. Absent SWCs during the first 72 h were also related to death. CONCLUSION: Early aEEG patterns can be predictive of neurological outcome in the preterm newborn. Low-voltage tracing and absence of SWCs are associated with severe neurological lesions/death.

Hepatocyte growth factor as an indicator of neonatal maturity.

Hepatocyte growth factor (HGF) is a potent mitogen and morphogen expressed in fetal organs. It requires activation by specific serine proteases. The role of activated HGF during fetal development has not been evaluated. We hypothesized that the serum levels of activated HGF would be increased in preterm neonates. Cord blood total HGF and activated HGF levels were measured in 19 preterm (gestational age, 28.04±2.39 weeks) and 24 term (gestational age, 39.37±0.95 weeks) newborns. Anthropometric parameters and metabolic indices were evaluated. Activated HGF was higher in preterm than in term neonates (0.81±0.05 vs. 0.61±0.06 ng/mL, p<0.05), whereas total HGF levels did not differ significantly between groups. In addition, total and activated HGF further increased by ∼40% in preterm neonates 12 h after birth. Finally, activated HGF correlated inversely with gestational age (r=-0.369; p=0.015) and birth weight (r=-0.440; p=0.003). Our study demonstrates that regulation of HGF activity and circulating HGF levels differ between term and preterm neonates along fetal development.

Inflammatory indices obtained from routine blood tests show an inflammatory state associated with disease progression in engineered stone silicosis patients.

Patients with silicosis caused by occupational exposure to engineered stone (ES) present a rapid progression from simple silicosis (SS) to progressive massive fibrosis (PMF). Patient classification follows international rules based on radiology and high-resolution computed tomography (HRCT), but limited studies, if any, have explored biomarkers from routine clinical tests that can be used as predictors of disease status. Our objective was thus to investigate circulating biomarker levels and systemic inflammatory indices in ES silicosis patients whose exposure to ES dust ended several years ago. Ninety-one adult men, ex-workers in the manufacturing of ES, 53 diagnosed with SS and 38 with PMF, and 22 healthy male volunteers (HC) as controls not exposed to ES dust, were recruited. The following circulating levels of biomarkers like lactate dehydrogenase (LDH), angiotensin-converting-enzyme (ACE), protein C reactive (PCR), rheumatoid factor, alkaline phosphatase and fibrinogen were obtained from clinical reports after being measured from blood samples. As biochemical markers, only LDH (HC = 262 ± 48.1; SS = 315.4 ± 65.4; PMF = 337.6 ± 79.3 U/L), ACE (HC = 43.1 ± 18.4; SS = 78.2 ± 27.2; PMF = 86.1 ± 23.7 U/L) and fibrinogen (HC = 182.3 ± 49.1; SS = 212.2 ± 43.5; PMF = 256 ± 77.3 U/L) levels showed a significant sequential increase, not been observed for the rest of biomarkers, in the HC → SS → PMF direction. Moreover, several systemic inflammation indices neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), platelet-to-lymphocyte ratio (PLR), systemic inflammation response index (SIRI), systemic immune-inflammation index (SII), aggregate index of systemic inflammation (AISI) derived from whole blood cell counts showed significant differences between the HC, SS and PMF groups. All these biomarkers were analyzed using receiver operating characteristic (ROC) curves, and the results provided moderately high sensitivity and specificity for discriminating between ES silicosis patient groups and healthy controls. Our study reveals that some inflammatory biomarkers, easily available from routine blood analysis, are present in ES silicosis patients even several years after cessation of exposure to ES silica dust and they could help to know the progression of the disease.

Respiratory adverse events during upper digestive endoscopies in children under ketamine sedation.

BACKGROUND: There is no evidence of the need for oxygen supplementation during upper digestive endoscopies under ketamine sedation in children, and the latest recommendations specifically state that it is not mandatory for the procedure. The aim of our study is to assess the incidence of respiratory adverse events during upper digestive endoscopies in children under Ketamine sedation when performed without oxygen supplementation, in accordance with the latest recommendations. METHODS: Eighty-eight children undergoing ketamine sedation for programmed upper digestive endoscopy at our Pediatric Intensive Care Unit were included. Patients needing other sedative agents different from ketamine were excluded. No patients received previous oxygen therapy. Suction equipment, oxygen, a bag-valve-mask, and age-appropriate equipment for advanced airway management were immediately available. The primary outcome measure was the incidence of desaturation episodes (i.e. FiO2 below 90% requiring an intervention). RESULTS: Fifty-five patients (62.5%) presented a desaturation episode during the procedure. Most desaturation episodes occurred during the endoscope introduction (78.2%), and 5 episodes were previous to the endoscope introduction (minute 0). Around sixty percent of patients (58.9%) required oxygen therapy and four patients required bag-mask ventilation. Once oxygen therapy was initiated, 34 patients (70.5%) required it during the complete procedure or part of it. CONCLUSIONS: Desaturation episodes occur frequently early on in the procedure. Our data suggest that the role of oxygen supplementation prior to, and during upper digestive endoscopies under ketamine sedation in children should be thoroughly assessed for future recommendations.

Differential epigenetic regulation between the alternative promoters, PRDM1α and PRDM1ß, of the tumour suppressor gene PRDM1 in human multiple myeloma cells.

Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. The transcription factor PRDM1 is a master regulator of plasma cell development and is considered to be an oncosuppressor in several lymphoid neoplasms. The PRDM1ß isoform is an alternative promoter of the PRDM1 gene that may interfere with the normal role of the PRDM1α isoform. To explain the induction of the PRDM1ß isoform in MM and to offer potential therapeutic strategies to modulate its expression, we characterized the cis regulatory elements and epigenetic status of its promoter. We observed unexpected patterns of hypermethylation and hypomethylation at the PRDM1α and PRDM1ß promoters, respectively, and prominent H3K4me1 and H3K9me2 enrichment at the PRDM1ß promoter in non-expressing cell lines compared to PRDM1ß-expressing cell lines. After treatment with drugs that inhibit DNA methylation, we were able to modify the activity of the PRDM1ß promoter but not that of the PRDM1α promoter. Epigenetic drugs may offer the ability to control the expression of the PRDM1α/PRDM1ß promoters as components of novel therapeutic approaches.

Transcriptional correlates of the pathological phenotype in a Huntington's disease mouse model.

Huntington disease (HD) is a fatal neurodegenerative disorder without a cure that is caused by an aberrant expansion of CAG repeats in exon 1 of the huntingtin (HTT) gene. Although a negative correlation between the number of CAG repeats and the age of disease onset is established, additional factors may contribute to the high heterogeneity of the complex manifestation of symptoms among patients. This variability is also observed in mouse models, even under controlled genetic and environmental conditions. To better understand this phenomenon, we analysed the R6/1 strain in search of potential correlates between pathological motor/cognitive phenotypical traits and transcriptional alterations. HD-related genes (e.g., Penk, Plk5, Itpka), despite being downregulated across the examined brain areas (the prefrontal cortex, striatum, hippocampus and cerebellum), exhibited tissue-specific correlations with particular phenotypical traits that were attributable to the contribution of the brain region to that trait (e.g., striatum and rotarod performance, cerebellum and feet clasping). Focusing on the striatum, we determined that the transcriptional dysregulation associated with HD was partially exacerbated in mice that showed poor overall phenotypical scores, especially in genes with relevant roles in striatal functioning (e.g., Pde10a, Drd1, Drd2, Ppp1r1b). However, we also observed transcripts associated with relatively better outcomes, such as Nfya (CCAAT-binding transcription factor NF-Y subunit A) plus others related to neuronal development, apoptosis and differentiation. In this study, we demonstrated that altered brain transcription can be related to the manifestation of HD-like symptoms in mouse models and that this can be extrapolated to the highly heterogeneous population of HD patients.

Altered plasma-type gelsolin and amyloid-ß in neonates with hypoxic-ischaemic encephalopathy under therapeutic hypothermia.

Hypoxic-ischemic encephalopathy (HIE) is a severe neonatal complication responsible for ∼23% of all neonatal deaths. Also, 30-70% of these patients will suffer lifetime disabilities, including learning impairment, epilepsy or cerebral palsy. However, biomarkers for HIE screening, or monitoring disease progression are limited. Herein, we sought to evaluate the clinical usefulness of plasma-type gelsolin (pGSN) and amyloid-beta (Aß) 40 and 42 as prognostic biomarkers for HIE. pGSN has been previously suggested as a feasible marker in other brain injuries and amyloid-beta 40 and 42 are classically assessed in neurodegenerative diseases. However, to our knowledge, they have not been previously assessed in HIE patients. We have analyzed plasma pGSN and Aß 40 and 42 levels in 55 newborns (16 controls, 16 mild and 23 moderate-severe HIE) at birth, during 72 h of therapeutic hypothermia, a gold-standard treatment for HIE, and 24 h after hypothermia. Aß levels were lower in HIE patients, and pGSN levels were progressively reduced in mild and moderate-severe HIE patients. The fact that pGSN reductions could predict the severity of HIE and significantly correlated with the time to undergo hypothermia supports the prognostic value of plasmatic pGSN. Further studies are warranted to investigate the role of pGSN in neonatal HIE.

Higher maturity and connective tissue association distinguish resident from recently generated human tonsil plasma cells.

Human plasma cells (PC) are present in cell suspensions obtained from the tonsil by mechanical disaggregation (PC(MECH)). The present study shows that a collagenase treatment of tonsillar debris remaining after mechanical disaggregation yielded similar proportions of PC (PC(COLL)). Moreover, PC(MECH) were present in suspensions highly enriched in germinal center cells whereas PC(COLL) contained most of the IgA-secreting cells, suggesting their predominant location in follicular and parafollicular areas and connective tissue-rich zones such as tonsil subepithelium, respectively. Tonsil PC(MECH) and PC(COLL) shared the phenotype CD38(high) CD19(+) CD20(low) CD45(high), expressed equivalent amounts of PRDI BF1/Blimp-1 transcription factor, and carried similarly mutated IgVH6 genes. However, they differed in several features. 1) PC(MECH) still expressed the early B cell transcription factor BSAP and were HLA-DR(high); in contrast, PC(COLL) were BSAP(-)and HLA-DR(low). 2) PC(MECH) were CD95(+) and Bcl-2(+/-) whereas PC(COLL) showed CD95(+/-) and Bcl-2(+) expression; in addition, PC(MECH) exhibited increased spontaneous apoptosis. 3) The two PC subsets exhibited distinctive adhesion molecule profiles, since PC(COLL) expressed higher levels of CD31, CD44, and CD49d, but a lower level of CD11a than PC(MECH). These results suggest that PC(MECH) are recently generated, short-living PC, and PC(COLL) constitutes a subset with higher maturity and survival, which resides in connective tissue-rich areas.

VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins.

The production and secretion of antibodies by human plasma cells (PCs) are two essential processes of humoral immunity. The secretion process relies on a group of proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which are located in the plasma membrane (t-SNAREs) and in the antibody-carrying vesicle membrane (v-SNARE), and mediate the fusion of both membranes. We have previously shown that SNAP23 and STX4 are the t-SNAREs responsible for antibody secretion. Here, using human PCs and antibody-secreting cell lines, we studied and characterized the expression and subcellular distribution of vesicle associated membrane protein (VAMP) isoforms, demonstrating that all isoforms (with the exception of VAMP1) are expressed by the referenced cells. Furthermore, the functional role in antibody secretion of each expressed VAMP isoform was tested using siRNA. Our results show that VAMP2 may be the v-SNARE involved in vesicular antibody release. To further support this conclusion, we used tetanus toxin light chain to cleave VAMP2, conducted experiments to verify co-localization of VAMP2 in antibody-carrying vesicles, and demonstrated the coimmunoprecipitation of VAMP2 with STX4 and SNAP23 and the in situ interaction of VAMP2 with STX4. Taken together, these findings implicate VAMP2 as the main VAMP isoform functionally involved in antibody secretion.

Monitoring of Blood Pressure is not Enough to Avoid Neonatal Postoperative Encephalopathy.

Background Neonatal encephalopathy with seizures after general anesthesia not occurring in infants undergoing cardiac or major neurosurgery is very uncommon. An ischemic origin due to cerebral hypoperfusion from perioperative hypotension has been suggested, but there is a lack of a consensus definition for intraoperatory hypotension in neonates. Case Report We report the first case of neonatal encephalopathy with seizures in a neonate with anorectal malformation. He underwent a colostomy with caudal anesthesia combined with light general anesthesia. Intraoperative systolic blood pressure and mean blood pressure values were considered normal. Thirty-two hours after the intervention, the patient presented electroclinical seizures. Diffusion-weighted imaging showed bilateral involvement with reduced diffusivity in the watershed areas of the middle cerebral artery and posterior cerebral artery. Conclusion Perioperative monitoring of blood pressure is not enough in neonatal surgery. Cerebral magnetic resonance imaging should be considered in infants with noncardiac congenital anomalies after neonatal surgery and long-term follow-up is required.

Prognostic Value of the Apparent Diffusion Coefficient in Newborns with Hypoxic-Ischaemic Encephalopathy Treated with Therapeutic Hypothermia.

BACKGROUND: Apparent diffusion coefficient (ADC) quantification has been proven to be of prognostic value in term newborns with hypoxic-ischaemic encephalopathy (HIE) who were treated under normothermia. OBJECTIVES: To evaluate the prognostic value of ADC in standardized brain regions in neonates with HIE who were treated with therapeutic hypothermia (TH). METHODS: This prospective cohort study included 54 term newborns who were admitted with HIE and treated with TH. All magnetic resonance imaging examinations were performed between days 4 and 6 of life, and ADC values were measured in 13 standardized regions of the brain. At 2 years of age we explored whether ADC values were related to composite outcomes (death or survival with abnormal neurodevelopment). RESULTS: The severity of HIE is inversely related to ADC values in different brain regions. We found that lower ADC values in the posterior limb of the internal capsule (PLIC), the thalami, the semioval centre, and frontal and parietal white matter were related to adverse outcomes. ADC values in the PLIC and thalami are good predictors of adverse outcomes (AUC 0.86 and 0.76). CONCLUSIONS: Low ADC values in the PLIC, thalamus, semioval centre, and frontal and parietal white matter in full-term infants with HIE treated with TH were associated with a poor outcome.

Modulated selection of IGHV gene somatic hypermutation during systemic maturation of human plasma cells.

Systemic antigen-induced PCs are generated in inductive lymphoid tissues. Some of them are selected to travel through the circulation and finally, to home onto BM niches. BM PCs show prolonged survival and secrete high-affinity antibodies. In this study, human PCs were isolated from tonsil, blood, and BM, their IGHV3 and IGHV6 genes were sequenced, and their SHM were evaluated. The SHM analysis reveals the existence of a maturational gradient in these genes, as demonstrated by a progressive increase in the frequency of total and R mutations and total and NC aa changes following the direction: tonsil --> blood --> BM. The ratio of R to S mutations in the CDR1 and -2, but not in the FRs, increases from tonsil to blood and BM; this parameter reaches a maximum threshold when more than 10 mutations/sequence occur. Further analyses indicate that CDR1 and CDR2 SHM followed different strategies to provide appropriate amino acid changes, but both exhibited maximal resistance to incorporating drastic molecular alterations in the BM PCs. Finally, all of the findings are similar in IGHV3 and IGHV6 sequences, indicating that they reflect general rules imposed by in vivo antigen selection.

Immunization-induced perturbation of human blood plasma cell pool: progressive maturation, IL-6 responsiveness, and high PRDI-BF1/BLIMP1 expression are critical distinctions between antigen-specific and nonspecific plasma cells.

The present study shows that reimmunization with tetanus toxoid (tet) caused a transient increase of the human blood plasma cell (PC) pool, detectable from 6th to 15th day postboost, as well as the temporal alteration of several PC features. Labeling of specific PC with FITC-tet C fragment (tetC) allowed kinetics analysis of the tetC(+) and tetC(-) PC, and revealed remarkable differences between them: 1) the kinetics of tetC(+) PC occurrence was exponential, and most of them appeared in a narrow time frame (5th to 8th day postboost), whereas the tetC(-) PC increase was lower (three to five times) and more prolonged (4th to 15th day postboost). 2) The tetC(+) PC subset contained a fraction of cycling cells, expressed high levels of DR, CD138, and CD126, and responded to IL-6 by improving their survival and Ig secretion; in contrast, the tetC(-) PC showed higher CXCR4 and lower DR and CD138, did not respond to IL-6, and contained a fraction of apoptotic cells. 3) Sequential phenotypic analysis revealed maturational changes within the tetC(+), but not tetC(-), PC subset; sequencing of tetC(+) PC IgVH genes showed clear features of Ag selection. 4) The tetC(+) PC expressed several times more positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1 transcription factor than the tetC(-) PC. 5) The tetC(-) PC and bone marrow resident PC similarly expressed low DR and high CXCR4, but differed in that the latter exhibited higher levels of CD31, CD138, and positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1. These findings support the view that tetC(+) PC contain bone marrow PC precursors, and tetC(-) PC probably belong to a removable compartment of aged PC.