PD-L2 expression extends beyond dendritic cells/macrophages to B1 cells enriched for V(H)11/V(H)12 and phosphatidylcholine binding

B1 B cells are the major source of natural antibody that is essential for innate immunity. The B1 repertoire is skewed toward production of phosphatidylcholine (PtC)-binding V(H)11 and V(H)12 immunoglobulin that plays a key role in immune defense against bacterial infection. Programmed death-ligand 2 (PD-L2) is a ligand for the immunosuppressive receptor programmed death-1 (PD-1). It has been reported that expression of PD-L2 is restricted to dendritic cells and macrophages in mice. Here we show that 50-70% of resting peritoneal B1 cells express PD-L2, which is not present or inducible on conventional B2 B cells or PD-L2(-) B1 cells. Although PD-L2(+) and PD-L2(-) B1 cells are similar in proliferative responses and spontaneous immunoglobulin secretion, PD-L2(+) B1 cells are highly enriched for expression of V(H)11 and V(H)12 genes and encompass the bulk of PtC-binding B1 cells. These findings extend the range of known PD-L2 expression to B cells and show that B1 cells identified by this marker express a specific repertoire associated with anti-bacterial immunity.     

Cancer mucosa antigens as a novel immunotherapeutic class of tumor-associated antigen

Colorectal cancer is a leading cause of cancer-related mortality worldwide. Surgery and chemoradiation exhibit incomplete efficacy and, ultimately, 50% of patients die of metastatic disease. In the context of that unmet clinical need, immunotherapeutic approaches have enjoyed limited success, partly because of a paucity of suitable antigen targets. However, exploitation of immune compartmentalization, employing antigens with expression restricted to normal intestinal mucosa and derivative colorectal tumors--cancer mucosa antigens (CMAs)--may represent a previously unrecognized class of immune targets supporting efficacious antitumor immunotherapy. Guanylyl cyclase C (GCC) is an intestine/colorectal cancer-restricted protein ideally suited as the first CMA for clinical evaluation.     

Management of a patient presenting with anterior STEMI with concomitant COVID-19 infection early in the course of the U.S. pandemic

The coronavirus disease-2019 (COVID-19) is a viral illness with heterogenous clinical manifestations, ranging from mild symptoms to severe acute respiratory distress syndrome and shock caused by the severe acute respiratory syndrome coronavirus-2. The global healthcare community is rapidly learning more about the effects of COVID-19 on the cardiovascular system, as well as the strategies for management of infected patients with cardiovascular disease. There is minimal literature available surrounding the relationship between COVID-19 infection and acute coronary syndrome. We describe the case of a woman who presented with an acute anterior ST-elevation myocardial infarction managed by primary percutaneous coronary intervention, who subsequently developed severe COVID-19 infection and ultimately succumbed to multisystem organ failure.     

Eleven novel mutations in the factor VIII gene from Brazilian hemophilia A patients

The molecular characterization of the mutations in hemophilia A patients is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protocol involving multiplex polymerase chain reaction in which 19 exons were amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutations. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) located in intron 22 of the factor VIII gene and two copies close telomeric region of X chromosome. Forty-two hemophilia A patients (21 with severe and 21 with mild-to-moderate disease) were studied. The inversion of factor VIII occurred in 13 of 21 patients affected by severe hemophilia A. One patient showed a large extra band in addition to the three bands observed after Southern blotting with the F8A probe. An abnormal electrophoretic pattern of SSCP was detected in 85% and 50% of the patients affected by mild-to-moderate and severe disease, respectively. Sixteen different mutations were identified. Eleven mutations were novel and comprised 9 point mutations and 2 small deletions. This study shows that the methodology used is safe and rapid and has potential for detecting almost all of the genetic defects of the studied hemophilia A patients.     

Down-modulation of neutrophil production by erythropoietin in human hematopoietic clones

In clonogenic assays of hematopoietic progenitors, high concentrations (4 U/mL) of erythropoietin (epo) reduced the formation of granulocyte- macrophage (GM) colonies and diminished the number of granulocytes formed per culture plate. Fetal progenitors were more sensitive to these effects of epo than were progenitors from adults, displaying these reductions at greater than or equal to 1 U epo/mL. The mechanism was investigated by growing fetal progenitors stimulated by recombinant GM-CSF, in the absence of epo, and when eight-cell clones first appeared, mapping their location, then adding epo, and assessing its effect on the subsequent differentiation of the clones. In the absence of epo, the clones developed exclusively into GM colonies. However, if developing clones were presented with epo, 85% matured into GM colonies, but 15% became multilineage or normoblast colonies. In addition, developing clones that were presented with epo produced colonies that contained fewer neutrophils. These effects of epo on neutrophil generation were observed with each of three varieties of recombinant epo, and also with purified human epo, but were not observed using epo that had been neutralized with rabbit anti-epo antiserum.     

Use of naloxone during cardiac arrest and CPR: potential adjunct for postcountershock electrical-mechanical dissociation

Naloxone has been shown to increase arterial pressure in hemorrhagic and septic shock. To determine if naloxone has salutary effects during cardiac arrest with conventional closed-chest cardiopulmonary resuscitation (CPR), ten dogs were studied during 20 minutes of ventricular fibrillation (VF) and CPR and during a 30-minute postcountershock period. Central aortic (Ao) and right atrial (RA) systolic and end-diastolic (EDP) pressures, instantaneous Ao-RA pressure difference (coronary perfusion pressure), and electromagnetic Ao flow were measured. Ao and RA samples were analyzed during a control period and at five-minute intervals during CPR for PO2, PCO2, and pH. During VF, a piston-cylinder device was used to perform anteroposterior sternal depressions and positive pressure ventilations (100% O2) at standard rates and ratios. After 15 minutes of CPR, animals were randomized and given either naloxone (5 mg/kg) or epinephrine (1 mg). Defibrillation was attempted five minutes later using 1 J/kg and then, if necessary, 2, 4, 8, 12, and 16 J/kg until VF was terminated or the maximum energy dose was reached. If VF persisted or if countershock resulted in asystole or a nonperfusing rhythm (electrical-mechanical dissociation [EMD]), the alternate drug (naloxone or epinephrine) was then given. Measured systolic pressures, coronary perfusion pressures, aortic flow, and blood gases were not significantly different during the control period or at five, ten, and 15 minutes of VF and CPR between animal groups prior to drug administration. When compared to hemodynamic values measured at 15 minutes, naloxone had no significant effect on pressures or aortic flow measured five minutes after administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Results of a phase I/II trial of recombinant human granulocyte- macrophage colony-stimulating factor in very low birthweight neonates: significant induction of circulatory neutrophils, monocytes, platelets, and bone marrow neutrophils

Neonates, especially those of very low birthweight (VLBW), have an increased risk of nosocomial infections secondary to deficiencies in development. We previously demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) production and mRNA expression from stimulated neonatal mononuclear cells are significantly less than that from adult cells. Recombinant murine GM-CSF administration to neonatal rats has resulted in neutrophilia, increased neutrophil production, and increased survival of pups during experimental Staphylococcus aureus sepsis. In the present study, we sought to determine the safety and biologic response of recombinant human (rhu) GM-CSF in VLBW neonates. Twenty VLBW neonates (500 to 1,500 g), aged < 72 hours, were randomized to receive either placebo (n = 5) or rhuGM-CSF at 5.0 micrograms/kg once per day (n = 5), 5.0 micrograms/kg twice per day (n = 5), or 10 micrograms/kg once per day (n = 5) given via 2-hour intravenous infusion for 7 days. Complete blood counts, differential, and platelet counts were obtained, and tibial bone marrow aspirate was performed on day 8. Neutrophil C3bi receptor expression was measured at 0 and 24 hours. GM-CSF levels were measured by a sandwich enzyme-linked immunosorbent assay at 2, 4, 6, 12, and 24 hours after the first dose of rhuGM-CSF. At all doses, rhuGM-CSF was well tolerated, and there was no evidence of grade III or IV toxicity. Within 48 hours of administration, there was a significant increase in the circulating absolute neutrophil count (ANC) at 5.0 micrograms/kg twice per day and 10.0 micrograms/kg once per day, which continued for at least 24 hours after discontinuation of rhuGM-CSF. When the ANC was normalized for each patient's first ANC, there was a significant increase in the ANC on days 6 and 7 at each dose level. By day 7, all tested doses of rhuGM- CSF resulted in an increase in the absolute monocyte count (AMC) compared with placebo-treated neonates. In those receiving rhuGM-CSF 5.0 micrograms/kg twice per day, there was additionally a significant increase in the day 7 and 8 platelet count. Tibial bone marrow aspirates demonstrated a significant increase in the bone marrow neutrophil storage pool (BM NSP) at 5.0 micrograms/kg twice per day and 10.0 micrograms/kg once per day. Neutrophil C3bi receptor expression was significantly increased 24 hours after the first dose of rhuGM-CSF at 5.0 micrograms/kg once per day. The elimination half-life (T1/2) of rhuGM-CSF was 1.4 +/- 0.8 to 3.9 +/- 2.8 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular characterization of a high A2 beta thalassemia by direct sequencing of single strand enriched amplified genomic DNA

Two families, one of Anglo-Saxon-Dutch descent, and the other, West Indian black, have an atypical beta thalassemia characterized by an unusually high level of Hb A2 in the heterozygous state. Restriction endonuclease mapping showed a deletion of about 1.35 kilobase (kb) in the 5' region of the beta globin gene. Direct sequencing of a specific region of genomic DNA amplified by a new modification of the polymerase chain reaction defined the deletion to be 1,393 base pairs (bp) and to be the same in both families. The deletion extends from 485 bp 5' to the mRNA CAP site to the middle of the second intervening sequence. This deletion, together with three others previously described that remove the 5' end of the beta gene but leave the delta gene intact, are all associated with unusually high levels of Hb A2 in the heterozygous state.     

Molecular analysis of relapse in chronic myeloid leukemia after allogeneic bone marrow transplantation

Four patients with Philadelphia (Ph') positive chronic myeloid leukemia (CML) were studied before, after, and on relapse following allogeneic bone marrow transplantation (BMT). Southern analysis of DNA from cells collected before and at relapse after BMT was performed in order to investigate the origin of the leukemia at relapse. Using minisatellite probes we showed that the relapse occurred in cells of host origin in all four patients and this was confirmed with a Y chromosome specific probe in two male patients who had a female donor. Furthermore, using two probes for the breakpoint cluster region (bcr) on chromosome 22, we showed that leukemic cells at relapse bore identical rearrangements to those in the disease at time of presentation of each patient. We conclude that relapse in all four patients is due to re-emergence of the original leukemic clone.