Endoreduplication in conjunction with tumor progression in an aneuploid laryngeal squamous cell carcinoma

We report the case of a 58-year-old man who presented with a squamous cell carcinoma pT1a G2 of the left vocal cord. Six months after histologically verified complete resection, the patient experienced an endolaryngeal and extralaryngeal local recurrence pT4 pN2b G2. We applied DNA flow cytometry (FCM) and comparative genomic hybridization (CGH) on both primary and recurrent tumor. The primary tumor and the endolaryngeal compartment of the relapse was an aneuploid cell clone with a FCM DNA index of 1.42 and 1.44, respectively. The extralaryngeal compartment showed a shift featuring a DNA index of 2.78. In the primary tumor and in both compartments of the recurrence there was an identical pattern of complex chromosomal imbalances as detected in CGH (CGH karyotype: rev ish enh [8q24.2-q24.3, 10q26.1-q26.3, 11q24-q25, 12q24.2-q23.33,X], dim [4q, 13q14.3-q31], amp[1p36.1-p36.2]). Hence, the recurrence was not associated with further gains and losses of chromosomal material. However, in the anterior part of the recurrence, the aneuploid tumor cell genome had completely doubled, obviously due to endoreduplication. Immunohistochemical analysis of several cell-cycle regulators revealed altered expression of checkpoint proteins, pointing to a complex disturbance in cell-cycle regulation.     

Diagnostic efficacy of two rapid tests for detection of respiratory syncytial virus antigen.

With the availability of ribavirin therapy for serious respiratory syncytial virus (RSV) infections, rapid diagnostic tests for the detection of RSV antigen are increasingly important. Efficacies of a commercially available enzyme immunoassay (EIA) (Abbott Laboratories, North Chicago, Ill.) and a fluorescent-antibody assay (FA) were evaluated in a study involving 135 specimens from children with respiratory symptoms. A nasal wash specimen was cultured immediately on RSV-sensitive A549 cells; the nasal wash was also used for EIA. FA was performed on a nasopharyngeal swab specimen with bovine anti-RSV and anti-bovine immunoglobulin G antisera (Burroughs Wellcome Co., Research Triangle Park, N.C.). A total of 39 specimens (28%) were tissue culture positive, including 35 EIA-positive and 37 FA-positive samples (sensitivities, 90 and 95%, respectively). All 96 tissue culture-negative specimens were EIA negative (specificity, 100%); 94 of these 96 specimens were FA negative (specificity, 98%). Positive and negative predictive values for the tests were as follows: 100 and 96% for EIA, respectively, and 95 and 98% for FA, respectively. Other viruses, including influenza A virus, adenovirus, enterovirus, and herpes simplex virus, were isolated in nine cases. One adenovirus-positive specimen had a false-positive RSV FA result; all nine specimens were RSV EIA negative. Both tests performed well in our study and provide cost-effective alternatives to tissue culture. The RSV EIA, in particular, uses standard serologic techniques and equipment and does not require expertise in virology. More widespread availability of rapid diagnostic tests for RSV will hopefully result in early and appropriate use of antiviral therapy in patients at risk for serious RSV infections.     

Coxsackievirus B3 replication and persistence in intestinal cells from mice infected orally and in the human CaCo-2 cell line

Although the transmission of coxsackievirus B3 occurs mainly via the oral route, little is known about the primary replication and persistence of this agent in the intestine. To address this question, BALB/c mice were inoculated by gavage with coxsackievirus B3, Nancy strain. The mice were killed from 1 hr to 90 days after infection. The viral markers were detected in the small intestine using RT-PCR, cell culture and detection of VP1 protein. Coxsackievirus B3 was detected positive by the three methods from hr 2 to day 45 after infection. By using monoclonal antibodies directed towards VP1, CD40 and CD26, the virus was shown to be present in the lymphocytes of the mucosa as soon as 2 hr after infection; in contrast, no virus was detected in the epithelial cells lining the intestinal lumen. Further experiments were performed to evaluate the capacity of coxsackievirus B3 to establish a persistent infection in two intestinal cell lines. In contrast to HT29 cells, the CaCo-2 cells were shown to develop a persistent infection for up to 20 passages, as demonstrated by the detection of viral RNA and VP1 protein. This study provides further evidence that, after infection by the oral route, the viral particles are concentrated in the lymphocytes of the mucosal layer. In addition, the results suggest that coxsackievirus B3 is capable of establishing a persistent infection in the small intestine that may act as a reservoir of viral particles for the delayed spread of the virus to other target organs.     

Nonmyeloablative bone marrow transplantation: Infectious complications in 65 recipients of HLA-identical and mismatched transplants.

Infections are a common complication of allogeneic bone marrow transplantation and the leading cause of transplantation-related mortality. It had been hypothesized that transplantation following nonmyeloablative preparative regimens would result in fewer infections by causing less mucosal injury, less graft-versus-host disease, and allowing earlier immune reconstitution. We have retrospectively reviewed the infectious complications of 65 consecutive patients with advanced hematologic malignancies who underwent bone marrow transplantation using a novel preparative regimen consisting of cyclophosphamide, thymic irradiation, and in vivo T-cell depletion. Cytomegalovirus (CMV) infection occurred in 52% of cases in which the donor or recipient had evidence of prior CMV exposure. Using a strategy of preemptive therapy and secondary prophylaxis with ganciclovir, no CMV disease occurred. Infections with gram-positive bacteria predominated over the first 100 days after bone marrow transplantation. Thereafter, the relative proportion of gram-negative infections increased without a significant increase in episodes of neutropenia. The rate of bacterial infections was not influenced by relapse of the underlying malignancy. Seven patients developed infections with Aspergillus species, which was the most common infectious cause of death in these patients. Infections with viruses other than CMV (n=10) and with protozoan organisms (n=2) also occurred. The use of HLA-mismatched donors, the occurrence of grade II-IV acute graft-versus-host disease, and treatment with corticosteroids did not influence the risk of CMV or bacterial or fungal infections in patients who underwent transplantation following this preparative regimen. Overall, the incidence and spectrum of infections in this series was similar to the reported incidence of infections following conventional myeloablative allogeneic stem cell transplantation. We conclude that a quantitative T-cell deficiency in these extensively T-cell depleted patients may be a risk factor for infection, even in the absence of graft-versus-host disease.     

Separate and variably shaped chromosome arm domains are disclosed by chromosome arm painting in human cell nuclei

Fluorescence in situ hybridization (FISH) with microdissection probes from human chromosomes 3 and 6 was applied to visualize arm and subregional band domains in human amniotic fluid cell nuclei. Confocal laser scanning microscopy and quantitative three-dimensional image analysis showed a pronounced variability of p- and q-arm domain arrangements and shapes. Apparent intermingling of neighbouring arm domains was limited to the domain surface. Three-dimensional distance measurements with pter and qter probes supported a high variability of chromosome territory folding.     

Aberrant chromatin at genes encoding stem cell regulators in human mixed-lineage leukemia

Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer.     

Pharmacological control of human basophil histamine release stimulated by eosinophil granule major basic protein.

Eosinophil granule major basic protein (MBP) is a 13,800 MW arginine-rich polypeptide that is unique among basic molecules in its ability to stimulate human basophil histamine release. We examined the Ca2+ requirements and pharmacological regulation of MBP-stimulated histamine release. Minimal MBP-induced histamine release occurred in the absence of extracellular Ca2+, whereas addition of 0.1 mM Ca2+ resulted in 70% of the maximum histamine release response. Maximum histamine release required 0.5 to 1 mM extracellular Ca2+. The MBP-induced histamine release was blocked by a calmodulin antagonist and by theophylline and was partially inhibited by an inhibitor of phospholipase A2. Release was unaffected by inhibition of protein kinase C. Basophil pretreatment with pertussis toxin also resulted in a concentration-dependent inhibition of release, suggesting involvement of a GTP regulatory protein in the activation mechanism. Histamine release stimulated by a 13,900 MW poly-L-arginine exhibited a dissimilar pharmacological profile from that of MBP. These results support the non-cytolytic nature of the MBP activation mechanism and identify pharmacological approaches for control of MBP-induced mediator release.     

Diastolic Blood Pressure and Heart Rate Biofeedback Training During Orthostatic Stress

Forty male college student volunteers were asked either to increase their diastolic pressure or their heart rate while sitting and during orthostatic stress (going from a sitting to a standing position), and half of them were also given second- to-second visual feedback for the target variable. Systolic blood pressure was also continuously recorded. Comparisons were made between baseline and voluntary control conditions, and test trials were included to examine immediate carry-over effects. With voluntary control instructions, substantial increases in tonic levels were obtained for the three cardiovascular variables in both sitting and postural change conditions. In general, the increases were significantly greater for feedback than for no-feedback conditions. Phasic effects of feedback were also observed during postural change conditions: the blood pressure troughs and the heart rate peak occurred earlier with feedback than for instructions only. Immediate transfer effects were obtained in feedback conditions only. The results were discussed in terms of concomitant effects of somatic and cognitive activities. The distinctive feedback effects on the time course of the responses suggest that a precise analysis of response patterns is needed in selecting appropriate feedback methods in the management of orthostatic hypotension.     

Host Defenses in Experimental Scrub Typhus: Delayed-Type Hypersensitivity Responses of Inbred Mice

Delayed-type hypersensitivity responses of inbred mice during the course of lethal and chronic infections with strains of Rickettsia tsutsugamushi were evaluated by using the influx of radiolabeled cells into antigen-injected ears. Congenic strains of C3H mice, which previously have been shown to be resistant (C3H/RV) or sensitive (C3H/HeDub) to lethal intraperitoneal infection with the Gilliam strain of rickettsiae, both expressed delayed-type hypersensitivity early in the course of infection (5 to 7 days). The sensitive C3H/HeDub mice, however, exhibited a marked decline in reactivity just before death. In contrast, reactivity of C3H/RV mice remained high through day 9 and declined slowly through day 15 after infection. Similar results were obtained when BALB/c mice were infected with either the Karp or the Gilliam strain of rickettsiae, which produce a lethal or nonlethal infection, respectively, in this strain of mice. Rechallenge of C3H/RV mice elicited a rapid increase in reactivity, suggesting a secondary memory response. To analyze delayed-type hypersensitivity during chronic infection, C3H/HeDub mice were immunized by subcutaneous infection with the Gilliam strain of R. tsutsugamushi, and both delayed-type hypersensitivity reactivity and resistance to intraperitoneal challenge were examined. Delayed-type hypersensitivity reactivity developed slowly and peaked at 21 days postimmunization, which correlated with resistance to intraperitoneal challenge. Delayed-type hypersensitivity reactivity declined thereafter, but resistance to intraperitoneal challenge remained through 28 days postimmunization. Delayed-type hypersensitivity reactivity increased after secondary challenge at 28 days, again suggesting antigen memory generated by primary immunization. Transfer of delayed-type hypersensitivity reactivity was accomplished by using immune thymus-derived splenic lymphocytes isolated with nylon-wool columns. Abrogation of the ability of immune spleen cells to transfer delayed-type hypersensitivity reactivity after treatment with anti-Thy 1.2 alloantiserum and complement further supported the view that delayed-type hypersensitivity responses to scrub typhus rickettsiae were mediated by thymus-derived lymphocytes.     

Irreversible neutrophil aggregation. A mechanism of decreased newborn neutrophil chemotactic response.

To investigate the neutrophil-neutrophil interactions of the newborn for possible clues to the etiology of decreased newborn neutrophil (PMN) chemotaxis, the authors compared adult and newborn C5a-induced PMN aggregation and chemotaxis at various PMN concentrations. Using Craddock's technique of C5a-induced aggregation, the authors found that the newborn lacks the normal biphasic aggregation-deaggregation seen in the adult, suggesting irreversible aggregation similar to that seen when adult PMNs are pretreated with cytochalasin-B. Chemotaxis of adult and newborn PMNs was studied with a modified Gallin radiolabel technique. A linear correlation between PMN concentration and corrected chemotactic response was found with both adult (r2 = 0.93) and newborn (r2 = 0.90) PMNs in the range 0.1 X 10(6) to 20 X 10(6) PMNs/ml. Random migration was not augmented by increased PMN concentration. The augmentation of newborn PMN chemotaxis was less than that of the adult (adult slope = 2426; newborn slope = 983). Irreversible newborn PMN aggregation may be the underlying event producing decreased PMN chemotaxis and interfering with the normal chemotactic augmentation caused by increased PMN concentration.