Increased Incidence of TdT-negative Pre-B Acute Lymphoblastic Leukemia Associated With Poor Prognostic Features Among Mexican Children in Central Mexico.

Mexican and Hispanic children in Mexico and the United States, respectively, have the highest incidence and worst outcomes of pre-B acute lymphoblastic leukemia (ALL) compared with other racial/ethnic groups. Terminal deoxynucleotidyl transferase (TdT) is an intranuclear DNA polymerase normally present on immature lymphocytes (TdT-positive) and distinguishes ALL from mature lymphoid malignancies. We performed a multisite retrospective study to determine the incidence of TdT-negative precursor B-cell acute lymphoblastic leukemia (pre-B ALL) among Mexican, Caucasian, and US-born Hispanic children to correlate TdT expression with patient characteristics and known prognostic factors. Fisher exact test was performed for categorical variables and the Wilcoxon rank-sum test was used for continuous variables. TdT-negative pre-B ALL was most frequently identified in patients with National Cancer Institute high-risk disease ( P =0.014). TdT-negative expression was also most frequently associated with hypodiploid pre-B ALL ( P =0.001) and KMT2A gene rearrangement ( P =0.0012). Mexican children had the highest incidence of TdT-negative ALL compared with Caucasians and US Hispanics ( P <0.001), with an increased incidence of poor prognostic features as well. This study demonstrates significant differences in TdT-negative expression, genomic alterations, and leukemic ploidy based on race and ethnicity.

P2X7 receptor blockade decreases inflammation, apoptosis, and enteric neuron loss during Clostridioides difficile toxin A-induced ileitis in mice.

BACKGROUND: Clostridioides difficile (C. difficile) is the most common pathogen causing health care-associated infections. C. difficile TcdA and TcdB have been shown to activate enteric neurons; however, what population of these cells is more profoundly influenced and the mechanism underlying these effects remain unknown. AIM: To characterize a specific population of TcdA-affected myenteric neurons and investigate the role of the P2X7 receptor in TcdA-induced ileal inflammation, cell death, and the changes in the enteric nervous system in mice. METHODS: Swiss mice were used to model TcdA-induced ileitis in ileal loops exposed to TcdA (50 µg/Loop) for 4 h. To investigate the role of the P2X7 receptor, Brilliant Blue G (50 mg/kg, i.p.), which is a nonspecific P2X7 receptor antagonist, or A438079 (0.7 µg/mouse, i.p.), which is a competitive P2X7 receptor antagonist, were injected one hour prior to TcdA challenge. Ileal samples were collected to analyze the expression of the P2X7 receptor (by quantitative real-time polymerase chain reaction and immunohistochemistry), the population of myenteric enteric neurons (immunofluorescence), histological damage, intestinal inflammation, cell death (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling), neuronal loss, and S100B synthesis (immunohistochemistry). RESULTS: TcdA upregulated (P < 0.05) the expression of the P2X7 receptor gene in the ileal tissues, increasing the level of this receptor in myenteric neurons compared to that in control mice. Comparison with the control mice indicated that TcdA promoted (P < 0.05) the loss of myenteric calretinin+ (Calr) and choline acetyltransferase+ neurons and increased the number of nitrergic+ and Calr+ neurons expressing the P2X7 receptor. Blockade of the P2X7 receptor decreased TcdA-induced intestinal damage, cytokine release [interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-α], cell death, enteric neuron loss, and S100B synthesis in the mouse ileum. CONCLUSION: Our findings demonstrated that TcdA induced the upregulation of the P2X7 receptor, which promoted enteric neuron loss, S100B synthesis, tissue damage, inflammation, and cell death in the mouse ileum. These findings contribute to the future directions in understanding the mechanism involved in intestinal dysfunction reported in patients after C. difficile infection.

Cutaneous B-Cell Lymphoblastic Lymphoma.

B-cell lymphoblastic lymphoma (B-LBL) is a malignant neoplasm of immature B cells that accounts for only 10% of all cases of lymphoblastic lymphoma. Most commonly, B-LBL presents as bony lesions, but in rare cases, the disease manifests cutaneously. We present a case of simultaneous cutaneous and systemic presentation of B-LBL in an otherwise healthy 28-year-old man in which the lymphoblastic infiltrate stained positive for CD79a, Tdt, CD10, and CD20. A diagnosis of cutaneous B-LBL was made, and systemic work-up revealed widespread involvement of the skin, bone, and lymph nodes. Review of all currently described cases of cutaneous B-LBL with or without systemic involvement revealed that the most frequently positive tumor markers were CD79a (92.3%), Tdt (90.6%), and CD10 (83.3%). Systemic involvement of B-LBL was found in nearly half of all cases with cutaneous presentation.

Posterior stromal cell apoptosis triggered by mechanical endothelial injury and basement membrane component nidogen-1 production in the cornea.

This study was performed to determine whether cells in the posterior stroma undergo apoptosis in response to endothelial cell injury and to determine whether basement membrane component nidogen-1 was present in the cornea. New Zealand White rabbits had an olive tip cannula inserted into the anterior chamber to mechanically injure corneal endothelial cells over an 8 mm diameter area of central cornea with minimal injury to Descemet's membrane. At 1 h (6 rabbits) and 4 h (6 rabbits) after injury, three corneas at each time point were cryopreserved in OCT for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemistry (IHC) for vimentin and nidogen-1, and three corneas at each time point were fixed for transmission electron microscopy (TEM). Uninjured corneas were controls. Stromal cells over approximately the posterior 25% of the stroma overlying to the site of corneal endothelial injury underwent apoptosis detected by the TUNEL assay. Many of these apoptotic cells were vimentin+, suggesting they were likely keratocytes or corneal fibroblasts. Stromal cells peripheral to the site of endothelial injury and more anterior stromal cells overlying the site of endothelial injury did not undergo apoptosis. Stromal cell death was confirmed to be apoptosis by TEM. No apoptosis of stromal cells was detected in control, uninjured corneas. Nidogen-1 was detected in the stroma of unwounded corneas, with higher nidogen-1 in the posterior stroma than the anterior stroma. After endothelial scrape injury, concentrations of nidogen-1 appeared to be in the extracellular matrix of the posterior stroma and, possibly, within apoptotic bodies of stromal cells. Thus, posterior stromal cells, likely including keratocytes, undergo apoptosis in response to corneal endothelial injury, analogous to anterior keratocytes undergoing apoptosis in response to epithelial injury.

Gradual strenuous running regimen predisposes to osteoarthritis due to cartilage cell death and altered levels of glycosaminoglycans.

OBJECTIVE: To investigate the hypothesis that strenuous running is a predisposing factor for osteoarthritis. DESIGN: Wistar rats were divided into two groups: a control group (CG) and a trained group (TG). The TG underwent a strenuous treadmill running training regimen of controlled intensity, exhibiting progressively improvement of fitness over 12 weeks, running at least 55 km during this period and finally performing an ultra-endurance running exercise to exhaustion. After this period, rats from both groups were euthanized and their knees removed. The articular cartilage was dissected and submitted to histomorphometrical, histomorphological, and immunohistochemical analyses evaluating cell death pathway (caspase-3 and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)) and inflammatory cytokines [interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α)]. In addition, the tissues were analyzed regarding the types and the content of glycosaminoglycans. RESULTS: The TG knee joints exhibited increase in the number of chondrocytes and chondrocyte clusters, as well as significantly increased levels of caspase-3, a protein involved in apoptosis, and of inflammatory cytokines IL-1α and TNF-α. In addition, histologically higher grades of osteoarthritis (Osteoarthritis Research Society International - OARSI grading), and significantly decreased levels of chondroitin sulfate and hyaluronic acid. Knee cartilage thickness and TUNEL did not significantly differ between the two groups. CONCLUSIONS: The articular cartilage of rats subjected to a strenuous running regimen of controlled intensity exhibited molecular and histological characteristics that are present in osteoarthritis.

B-cell lymphoblastic lymphoma in the maxilla of a child: a rare case report.

Lymphoblastic lymphoma is a malignant neoplasia that originates from B or T lymphocyte precursors and rarely occurs in the mouth. The authors report a rare case of B-cell lymphoblastic lymphoma in the maxilla of a child. Clinical examination revealed facial asymmetry with a swelling of the right maxilla, covered by healthy mucosa and painful to palpation. Radiographic examination revealed a poorly defined radiolucent lesion. Based on the hypothesis of malignant neoplasia of hematopoietic origin, an incisional biopsy was performed. Histological examination revealed malignant neoplasia with proliferation of monomorphic, lymphoid cells. Immunohistochemical staining was positive for leucocyte common antigen (LCA), CD10, CD20, CD79, and terminal deoxynucleotidyl transferase (TdT). After the diagnosis of B-cell lymphoblastic lymphoma, the patient underwent chemotherapy, but died of leukoencephalopathy and demyelinization caused by high doses of methotrexate.

Highly restricted diversity of TCR delta chains of the amphibian Mexican axolotl (Ambystoma mexicanum) in peripheral tissues.

Gammadelta T cells localize at mammalian epithelial surfaces to exert both protective and regulatory roles in response to infections. We have previously characterized the Mexican axolotl (Ambystoma mexicanum) T cell receptor delta (TRD) chain. In this study, TRD repertoires in spleen, liver, intestine and skin from larvae, pre-adult and adult axolotls were examined and compared to the thymic TRD repertoire. A TRDV transcript without N/D diversity, TRDV1S1-TRDJ1, dominates the TRD repertoires until sexual maturation. In adult tissues, this canonical transcript is replaced by another dominant TRDV1S1-TRDJ1 transcript. In the thymus, these two transcripts are detected early in development. Our results suggest that gammadelta T cells that express the canonical TRDV1S1-TRDJ1 transcript emerge from the thymus and colonize the peripheral tissues, where they are selectively expanded by recurrent ligands. This particular situation is probably related to the neotenic state and the slow development of the axolotl. In thymectomized axolotls, TRD repertoires appear different from those of normal axolotls, suggesting that extrathymic gammadelta T cell differentiation could occur. Gene expression analysis showed the importance of the gut in T cell development.

Maturation-associated immunophenotypic abnormalities in bone marrow B-lymphocytes in myelodysplastic syndromes.

Recent studies concerning the pathophysiology of myelodysplastic syndromes (MDS) have shown evidences for the existence of complex interactions between hematopoietic stem cells and the bone marrow (BM) microenvironment. We analyzed the B-lymphocyte maturation in BM of patients with MDS. For this purpose, 41 newly-diagnosed patients were analyzed. Enumeration and characterization of CD34+ and CD34- B-cell precursors and mature B-lymphocytes was performed using multiparameter flow cytometry. BM from eight transplant donors and six orthopedic surgery patients were used as controls. CD34+/CD45(lo) B-cells were found in 17/22 patients with RA/RARS and in 5/13 with RAEB. In patients with RAEB-t and CMML no CD34+ B-cell precursors could be detected. A positive correlation was found between CD34+ and CD34- B-cell precursors (r=0.52). CD34+ B-cell precursors presented an inverse correlation with BM percentage of blasts and peripheral leukocytes and a positive one with hemoglobin. Asynchronous antigen expression (CD19+/CD79a- cells) was found in 7/11 cases of RA/RARS and 6/18 cases of RAEB in which this phenotype was examined. Abnormal patterns of expression for at least one antigen was found in 91% of RA/RARS cases and in 74% of RAEB. Underexpression of TdT and CD79a were the most frequent abnormalities. Our results present evidences of an abnormal B-cell maturation in MDS. This may be an evidence that B-lymphocytes are derived of the abnormal clone. But it may also be the consequence of influences of abnormalities of BM microenvironment leading to an impaired commitment and maturation of the B-cell line in MDS. Studies performed with purified well-characterized B-cells may further elucidate these abnormalities.

Impact of shock and fluid resuscitation on the morphology and apoptosis of bone marrow: an experimental study.

BACKGROUND: We hypothesized that bone marrow failure after hemorrhagic shock might be secondary to impaired apoptosis regulation. Our objective was to assess the morphologic alterations and the rate of apoptosis in bone marrow after hemorrhagic shock and resuscitation. METHODS: Under pentobarbital anesthesia, Wistar rats (n = 70) underwent femoral vessel cannulation. The hemorrhagic shock model involved a controlled retrieval of blood, maintaining mean blood pressure at 40 +/- 5 mm Hg during 50 minutes. During the resuscitation period, lactated Ringer's (twice the blood volume retrieved, group LR) or NaCl 7.5% (4 mL/kg, group HS) was infused, followed by the previously retrieved blood. Bone marrow was collected through left femoral puncture. Morphology was assessed by Leishmann-stained smears, and apoptosis was assessed through terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay. Analysis of variance and Tukey's test were applied for statistical treatment, considering p < 0.05 as significant. RESULTS: LR animals presented a statistically significant decrease in the lymphocytic series (LR, 24.2 +/- 4.2%; Sham, 55.1 +/- 6.6%), together with an increase in the percentage of granulocyte (LR, 51.4% +/- 2.3%; Sham, 31.5 +/- 2.9%) and monocyte precursors (LR, 7.3 +/- 1.3%; Sham, 3.3 +/- 1.1%), detected 72 hours after shock (p < 0.05). Both LR and HS groups presented a significant increase in apoptosis, when compared with the sham group (LR, 13.1 +/- 0.5%; HS, 12.2 +/- 0.7%; Sham, 6.8 +/- 0.4%). The alterations detected in the bone marrow morphology of LR group were not observed in HS animals. CONCLUSION: There was an increase in bone marrow apoptosis after hemorrhagic shock. The type of resuscitation scheme used did influence bone marrow morphology.

Early expression of two TdT isoforms in the hematopoietic system of the Mexican axolotl. Implications for the evolutionary origin of the N-nucleotide addition.

Nontemplate (N)-nucleotide addition by the terminal dideoxynucleotidyl transferase (TdT) at the junctions of rearranging V( D) J gene segments greatly contribute to antigen-receptor diversity. TdT has been identified in several vertebrate species, where it is highly conserved. We report here the isolation of two forms of TdT mRNA in an amphibian, the Mexican axolotl. The isoform TdT1 shares all of the conserved structural motifs required for TdT activity and displays an average of 50-58% similarity at the amino acid level with TdT of other species. The second axolotl TdT variant ( TdT2) differs from TdT1 by a 57-amino acid deletion located between amino acids 165-222 of TdT1, including the first helix-hairpin-helix DNA-binding motif. During ontogeny, TdT products are first detected in the head of 6-week-old larvae and further in the head and trunk of 8-month-old larvae. These developmental stages correspond to the first detection of RAG1 and antigen-receptor (TCRbeta and IgHmicro) products in axolotl larvae. Our results suggest that in contrast to mammalian development, N diversity occurs early in axolotl development to diversify the primary repertoire. Phylogenetic analyses reveal that TdT and DNA polymerase mu(Pol mu) genes are closely related, and that both enzymes were already present in the common ancestor of jawed vertebrates.

Immunocytochemical detection of DNA and RNA in endosymbiont-bearing trypanosomatids.

Research about the kinetoplast of trypanosomatids has yielded valuable information about the organization of extranuclear structure. However, the ultrastructural localization of nucleic acids within these protozoa remains uncertain. We have applied cytochemical and immunocytochemical approaches to precisely identify DNA and RNA in lower endosymbiont-bearing trypanosomatids. Using the Terminal deoxynucleotidyl Transferase (TdT) immunogold technique, we showed that nuclear DNA is seen associated with the nuclear envelope during the trypanosomatid cell cycle. By combining the TdT technique with the acetylation method, which improves the contrast between structures containing fibrils and granules, we have demonstrated that the nucleolus of endosymbiont-bearing trypanosomatids is composed of two constituents: a granular component and a DNA-positive fibrillar zone. Moreover, we revealed that DNA of endosymbiotic bacteria consisted of electron-dense filaments which are usually in close contact with the prokaryote envelope. Using a Lowicryl post-embedding immunogold labeling procedure with anti-RNA antibodies, we showed the presence of RNA not only over the cytoplasm, the interchromatin spaces and the nucleolus, but also over the kinetoplast and virus-like particles present in Crithidia desouzai.

Apoptotic cell death is an important cause of neuronal injury in experimental Venezuelan equine encephalitis virus infection of mice.

Mice develop a fatal encephalomyelitis after infection with the Trinidad donkey strain of Venezuelan equine encephalitis (VEE) virus. Adult mice were inoculated intraperitoneally with VEE virus and the brains were examined at different time points. Morphological changes were assessed by histological staining. VEE virus antigen was detected with immunoperoxidase staining, and DNA fragmentation was evaluated in situ using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. VEE antigen was found in many areas of the brain and it was prominent in neurons. There were mild associated inflammatory changes. DNA fragmentation was demonstrated in many of these areas using TUNEL. In areas with TUNEL staining, morphological neuronal changes ranged from nuclear chromatin condensations to nuclear and cellular fragmentation, which are characteristic of apoptosis. There is strong morphological and biochemical evidence of apoptotic cell death in this experimental model of VEE virus infection.

Detection of TdT-positive cells in the mesenteric lymph nodes of immunodeficient rats: study of their antigenic phenotype.

Terminal deoxynucleotidyl transferase (TdT) containing cells were found in the mesenteric lymph nodes of protein deprived and casein re-fed rats. Double immunofluorescence was used to characterize these TdT+ cells according to their surface antigenic phenotype. TdT+ cells expressing T-cell antigen markers recognized by monoclonal antibodies: W3/13 and OX-19 indicated thymic origin. It was found that these cells represented half the existing TdT+ population in the mesenteric lymph nodes. The rest of them presented the Ia antigen which is coded for by the class II major histocompatibility complex and is recognized by the OX-6 mAb. TdT+ cells presenting the OX-6+ phenotype were ascribed to a bone marrow derived subset. These results indicate that, in some instances, i.e., immunodeficiency due to protein deprivation, TdT+ cells may appear in the mesenteric lymph nodes. Their origin may be attributed either to trafficking of immature cells from the thymus or to cells that leave the bone marrow as a consequence of the damage provoked by protein deprivation.

Biphenotypic leukemia: immunologic and morphologic evidence for a common lymphoid-myeloid progenitor in humans.

A 5-year-old white girl had a white blood cell count of 80,000/cu mm and 82% lymphoblasts in the peripheral blood. Acute lymphocytic leukemia (ALL) was diagnosed, defined by typical morphology (FAB-L1), a positive reaction for terminal deoxynucleotidyl transferase (TdT) and characteristic surface antigens detected with lymphoid monoclonal antibodies. The patient's peripheral lymphoblast count fell rapidly with ALL therapy, but the WBC count began to rise unexpectedly on the sixth day of treatment, with 84% myeloblasts and monocytoid blasts. Malignant cells in the bone marrow showed FAB-M4 morphology and were no longer reactive with antibodies directed against TdT or common ALL antigen. However, the myeloblasts continued to react with some of the same monoclonal antibodies as the original leukemia cells, and in addition expressed new determinants detected by monoclonal antibody TA-1 and peanut agglutinin. The rapid dynamic evolution of the malignancy during the course of induction chemotherapy favors the existence of a stem cell capable of differentiation into both lymphoid and myeloid clones that are both independently and selectively sensitive to specific chemotherapeutic regimens.