A case of aggressive multiple myeloma with cleaved, multilobated, and monocytoid nuclei, and no serum monoclonal gammopathy.

Multiple myeloma is a B-cell malignancy characterized by proliferation of neoplastic plasma cells. A few cases have been reported identifying variant forms of neoplastic plasma cells with atypical nuclei that secrete myeloma protein. We report a highly unusual case of plasma cell myeloma that presented with cleaved, multilobated, and monocytoid nuclei, without detectable myeloma protein in the serum or urine. The bone marrow contained sheets of plasma cells exhibiting pleomorphic nuclei with cleaved, multilobated, and monocytoid features that were negative for myeloperoxidase and dual esterase. Flow cytometric analysis revealed CD38high/CD45low cells expressing cytoplasmic kappa light chain, without evidence of myeloid or lymphoid differentiation. Following chemotherapy, the patient developed secondary plasma cell leukemia. A high plasma cell labeling index was obtained from bone marrow and peripheral blood, indicating a poor prognosis. In addition to quantitative immunoglobulins, serum protein electrophoresis, and immunofixation electrophoresis of serum and urine, we recommend cytochemical and flow cytometric studies for evaluation of suspected plasma cell myeloma with atypical cellular features.

Acute exposure to the abused inhalant, isobutyl nitrite, reduced T cell responsiveness and spleen cellularity.

Isobutyl nitrite is an inhalant abused principally by male homosexuals. We have reported that subchronic inhalation exposure (45 min/day for 14 days) to 900 ppm isobutyl nitrite was immunosuppressive. In the present study, the effects of acute exposure to the inhalant were examined. Mice were exposed in an inhalation chamber to 900 ppm isobutyl nitrite for 45 min. One day later, spleen cellularity was reduced by 39% without selectively depleting CD4(+) or CD8(+) cells. The numbers of peripheral blood leukocytes and peritoneal cells were also reduced. Following acute inhalation exposure, T cell proliferative responses stimulated with allogeneic cells or anti-CD3 and anti-CD28 antibodies were inhibited, while mitogen-induced responses were not affected. Purified T cells exposed to the inhalant also had compromised responses, suggesting a direct effect on T cells. However, the cumulative effects of multiple exposures were necessary to inhibit T-dependent antibody responses or T cell or macrophage cytotoxicity.

Nitrite inhalants spontaneously liberate nitric oxide, which is not responsible for the immunotoxicity in C57BL/6 mice.

Nitrite inhalant abuse has been correlated epidemiologically with HIV seropositivity and with Kaposi's sarcoma. Using a mouse model, we have shown that inhaled isobutyl nitrite caused anemia and severely depressed immunity. In the present study, we showed that both isobutyl and cyclohexyl nitrites in air liberated nitric oxide (NO). An immunotoxic dose of 900 ppm isobutyl nitrite liberated 115 ppm NO. Mice were exposed in an inhalation chamber to 115 ppm NO, 900 ppm isobutyl nitrite, or 900 ppm cyclohexyl nitrite for 45 min/day. Following a single exposure, NO did not affect peripheral blood cell counts, while isobutyl and cyclohexyl nitrites reduced cell numbers. After 14 daily exposures, isobutyl nitrite, but not cyclohexyl nitrite or NO, reduced peritoneal macrophage tumoricidal activity. The nitrite esters likely caused immunotoxicity by mechanisms other than NO release.

Severe hemorrhagic complication due to acquired factor V inhibitor after single exposure to bovine thrombin product.

Hemorrhagic complications have been reported after repeated exposures to bovine thrombin products due to development of factor V inhibitors. Our patient underwent emergency repair of acute aortic dissection and coronary artery bypass grafting. The patient developed leg wound infection at the saphenous vein harvest site, which was debrided and left open. Attempt to reclose the leg wound 1 month later was complicated by a life-threatening hemorrhage with markedly elevated activated partial thromboplastin time. There was no evidence of infection or disseminated intravascular coagulation, and further study identified low factor V level with positive factor V inhibitor. Treatment with plasmapheresis and steroid successfully reversed the coagulopathy. Detailed case review failed to reveal exposure to any thrombin products other than the one used for the aortic dissection repair. This case was unusual because only a single exposure to this product resulted in severe hemorrhagic complication 1 month after surgery.

Adeno-associated virus Rep78 inhibits oncogenic transformation of primary human keratinocytes by a human papillomavirus type 16-ras chimeric.

Seroepidemiologic studies demonstrate that adeno-associated virus (AAV) infection is negatively associated with cervical cancer. This inverse association may be due to an ability of AAV to inhibit the role of human papillomavirus (HPV) in cervical carcinogenesis. In support of this hypothesis AAV has been demonstrated to inhibit several papillomavirus types, including bovine papillomavirus type 1 and human papillomaviruses types 16 and 18 (HPV-16/18) in tissue culture. The AAV-encoded Rep78 protein was responsible for this inhibition. These previous studies, however, were largely carried out in immortalized mouse fibroblasts. This cell type is likely not to be the most accurate model cell type for studying HPV-associated cervical carcinogenesis. In this study it is demonstrated that AAV Rep78 protein inhibits oncogenic transformation of freshly explanted primary human foreskin keratinocytes by an HPV-16/ras chimeric genome. Such cells are the natural host cell type for HPV-16/18 infection. It is also demonstrated that the HPV-16 P97 promoter is specifically inhibited by Rep78 in a transient CAT assay. These data further extend our knowledge of the AAV-papillomavirus interaction and provide a model for investigating the negative association of AAV with cervical cancer.

Quantification of vitamin D receptor mRNA by competitive polymerase chain reaction in PBMC: lack of correspondence with common allelic variants.

It has been recently claimed that polymorphism for the vitamin D receptor (VDR) influences several aspects of calcium and bone metabolism. To evaluate the physiologic plausibility of these claims, we compared the abundance of the VDR mRNA in peripheral blood mononuclear cells (PBMCs) between different VDR genotypes using a quantitative reverse transcribed polymerase chain reaction-based method. The method is based on the coamplification of VDR cDNA and an internal standard consisting of known concentrations of a human VDR CDNA mutated at a BglII restriction site; the interassay coefficient of variation is 11%. To validate the method, we made use of earlier receptor binding studies indicating that normal human monocytes and activated, but not resting, lymphocytes expressed the VDR. The concentration of the VDR mRNA was 10(-8) to 10(-7) g/g of total RNA in cell-sorted monocytes and in in vitro activated lymphocytes, but only 10(-12) g/g of total mRNA in resting lymphocytes, establishing that the VDR mRNA determined by our method in PBMCs is due to constitutive expression in monocytes. Following an initial genotype screening of 85 normal volunteers by polymerase chain reaction or restriction fragment length polymorphism analysis, 14 individuals with the Bb genotype, 12 with the bb genotype, and 12 with the BB genotype were selected. The concentration of the VDR mRNA, corrected for the number of monocytes, was similar among the three genotype groups, as were the other variables examined: serum calcitriol, serum osteocalcin, and vertebral and hip bone density. We conclude that VDR polymorphism does not affect the abundance of the VDR mRNA.

Acute blood toxicity of the abused inhalant, cyclohexyl nitrite.

Cyclohexyl nitrite is an abused nitrite inhalant. This is the first report of toxicity of cyclohexyl nitrite. Mice were exposed to 300-900 ppm cyclohexyl nitrite in an inhalation chamber for 45 min and then bled. Such treatment resulted in a 7-10% reduction in red blood cell counts, haemoglobin and haematocrit levels. Both blood leucocyte counts and spleen cellularity were reduced by 40%. Unlike isobutyl nitrite, subchronic treatment of mice with cyclohexyl nitrite did not impair macrophage tumoricidal activity or production of reactive nitrogen intermediates, but did modulate B and T cell mitogen responses. The data suggest that cyclohexyl nitrite had cytotoxic activity, comparable to that of isobutyl nitrite, which might be related to the anaemia reported in abusers. The immunomodulatory properties of cyclohexyl nitrite differed from those of isobutyl nitrite.

Preceding standard therapy is the likely cause of MDS after autotransplants for multiple myeloma.

Myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) have been reported after autologous transplantation (AT) for lymphoma. It is not clear whether myeloablative therapy used in conjunction with autologous transplantation contributes to the development of MDS/AML or whether the conventional chemotherapy preceding the transplant, and administered over a prolonged period, causes these secondary malignancies. To address this issue, we examined 188 patients with multiple myeloma (MM) who had received AT. 71 patients with no more than one cycle of standard chemotherapy were enrolled in our Total Therapy program, designed to avoid exposure to alkylating agents prior to peripheral blood stem cell mobilization (group 1). The median duration of pretransplant therapy in group 1 was 7.6 months and significantly shorter than the 24 months of 117 patients (group 2) with more prolonged conventional therapy (P = 0.0001). All seven patients developing MDS post-transplantation belonged to group 2 (P = 0.02); the median durations from initial therapy and first transplant were 66 months (range 38-86) and 24 months (range 9-39), respectively. Our findings provide evidence that prolonged standard-dose alkylating agent therapy prior to transplantation, rather than autotransplant-supported myeloablative treatment, is associated with development of MDS/AML. Stem cell damaging alkylator treatment should be avoided, not to compromise PBSC collection, but also to reduce the risk of treatment-related MDS/AML.

Leukopenia and altered hematopoietic activity in mice exposed to the abused inhalant, isobutyl nitrite.

Isobutyl nitrite is representative of a group of inhalants abused primarily by male homosexuals; abuse of this drug may be a risk factor for AIDS or Kaposi's sarcoma. Using a 14-day exposure regimen, we previously reported that inhaled isobutyl nitrite was immunotoxic to mice, severely compromising T-dependent antibody responses and cytotoxic T cell and macrophage tumoricidal activity. In addition, exposure to the inhalant dramatically reduced spleen cellularity. A single 45-minute inhalation exposure produced anemia in mice. In the present study, we examined the effects of subchronic exposure to the drug on peripheral blood cellularity and hematopoietic activity. Mice were exposed to 900 ppm isobutyl nitrite in an inhalation chamber for 45 minutes/day for 14 days. One day after the final exposure, the number of peripheral blood leukocytes was reduced by 32%; however, the number of erythrocytes was increased by 7%. This was accompanied by an apparent shift from myelopoiesis to erythropoiesis. The numbers of bone marrow and spleen burst-forming units-erythroid (BFU-E) were increased about two-fold, while the numbers of colony-forming units-granulocyte/macrophage (CFU-GM) were decreased by about half. Bone marrow stromal cells also had reductions in the production of myeloid colony-stimulating activity after subchronic exposure to the inhalant. In addition, the numbers of hematopoietic stem cells, colony-forming units-spleen (CFU-S), were reduced in both bone marrow and spleen. Peripheral blood erythrocyte and leukocyte counts returned to normal levels by 7 days after the final exposure, as did the number of BFU-E. The number of CFU-GM remained depressed, however, even after 7 days of recovery. These data suggest that repeated exposures nonspecifically depleted cells and that erythropoiesis was stimulated, apparently at the expense of myelopoiesis.

Acute inhalation exposure to isobutyl nitrite causes nonspecific blood cell destruction.

Abuse of nitrite inhalants is widespread among male homosexuals and has been epidemiologically correlated with seropositivity to human immunodeficiency virus (HIV) and to Kaposi's sarcoma. These drugs may act as cofactors in AIDS if they compromise the ability to resist infection or tumor growth. We have previously reported that 14 daily 45-minute exposure to 900 ppm isobutyl nitrite in an inhalation chamber did compromise the immunocompetence of mice. We now report that a single 45-minute exposure produced a transient anemia. Erythrocyte counts, hemoglobin, and hematocrit levels were reduced by 7% but rebounded to above-normal levels 24 hours later. In vitro exposure of blood to isobutyl nitrite vapors did not lyse the cells but did induce Heinz body formation and increase their binding to macrophages. Thus, it is likely that the red cells were removed by phagocytic clearance not by direct lysis. Blood leukocyte numbers were also reduced following a single exposure to the inhalant, but the cell loss was delayed until 24 hours after exposure. Recovery of peripheral blood leukocytes 72 hours after exposure coincided with a reduction in spleen cellularity, suggesting that spleen cells were mobilized to replace lost blood leukocytes.

Reticulocyte counting by flow cytometry. A comparison with manual methods.

The reticulocyte count (RC) is a key diagnostic test in the evaluation, classification, and response to therapy of anemia. The RC, as determined by manual methods, has a frustrating inherent imprecision owing to its binomial counting statistics (i.e., low counts/low precision) and inaccuracy because of inter- and intraobserver variability as to what indeed is a reticulocyte. Fluorescent activated cytometric (FACS) analysis of reticulocytes by thiazole orange (TO) is a rapid, relatively simple, and precise method for counting reticulocytes. The automated method counts 10,000 cells or more vs. 1,000 cells counted by the manual method. Although inherently more precise, the FACS method may be inaccurate owing to the presence of Howell-Jolly bodies, nucleated red blood cells (RBCs), sickled cells, or giant platelets. The RC by FACS is well correlated with the manual method and the reference ranges are similar. A new parameter by FACS, the reticulocyte maturation index (RMI), provides an independent measurement of reticulocyte RNA content. Although the RMI does not correlate with RC either by FACS or manual methods, it does provide an independent parameter of erythropoietic activity and may be useful in predicting bone marrow engraftment or further subclassifying anemias. Determination by FACS of the RC offers significant advantages over manual methods in monitoring a patients erythropoietic response. However, one must be cognizant of potential pitfalls in the method.

Platelet dysfunction in Noonan's syndrome. A case with a platelet cyclooxygenase-like deficiency and chronic idiopathic thrombocytopenic purpura.

Individuals with Noonan's syndrome are likely to have one or more coagulation abnormalities: complex platelet function defects, partial Factor XI deficiency, or von Willebrand's disease. A distinctive platelet function defect has not been identified. The authors describe a 24-year-old women with Noonan's syndrome, chronic idiopathic thrombocytopenic purpura (ITP), and a platelet function defect characterized by a greater than 15-minute bleeding time, failure of aggregation and release with 10 microM ADP, 10 microM epinephrine, 750 microM arachidonic acid or 0.019 g/L collagen. A mixture of aspirin-treated platelets with the patient's platelets failed to correct the defect. Addition of 2.5 microM U46619 (a PGG2 analogue) corrected the aggregation and release defect. An electron microscopic analysis failed to reveal structural abnormalities. Thus, the platelet function defect in this patient appears to be a functional deficiency of cyclooxygenase. The presence of autoantiplatelet antibodies in a clinical setting consistent with chronic ITP raises the possibility that the defect may be acquired.

The use of pentoxifylline in a pig random skin flap model.

Pentoxifylline, the first drug in a new class of rheologic agents, has enhanced skin flap survival in previous rat models. In an attempt to find an animal model more similar to the human, a trial using a pig skin flap model was undertaken. Experimental animals received oral pentoxifylline for 21 days. Random skin flaps were elevated on the 14th day, and flap viability was determined on the 21st day. Similar flaps were raised on control animals. Serum fibrinogen levels, platelet aggregation studies, and erythrocyte flexibilities were measured in both groups. No improvement in flap viability or rheologic properties of the blood were shown in the pentoxifylline-treated animals. Although no enhancement of skin flap survival was demonstrated, future studies are warranted. Implications for future investigation and clinical application are discussed.

The role of apheresis in the support of life-threatening ITP relapse.

A 14-year-old girl with chronic idiopathic thrombocytopenic purpura (ITP) presented in relapse with a platelet count of 1,000/microL and a high-level serum antiplatelet IgG antibody. She previously had been unresponsive to courses of therapy with steroids, vincristine, and splenectomy. When treatment with danazol and purified immunoglobulins was unsuccessful in controlling her rapidly progressive course, an 8-day plasma exchange procedure was initiated in combination with platelet transfusion therapy and immunosuppression with cyclophosphamide and vincristine. Within 2 days, her clinical state improved markedly, correlating with a drop in her serum antiplatelet antibody level. She continued to improve and was discharged on a regimen of cyclophosphamide and danazol. Her antiplatelet antibody level had fallen to within the normal range, despite a typical platelet count of 5,000/microL during the 8-day period. Two weeks later her platelet count rose to 65,000/microL. This case suggests that a course of therapeutic plasma exchange may have a temporizing role in the acute management of life-threatening chronic ITP relapse, generating time for the more definitive therapy of immunosuppression to take effect.

Micrococcal nuclease as a DNA structural probe: its recognition sequences, their genomic distribution and correlation with DNA structure determinants.

We have analyzed micrococcal nuclease (MNase) DNA cleavage patterns at the sequence level by examining 2.3 X 10(3) base-pairs of data derived from the Drosophila melanogaster 44D larval cuticle locus. Within this region, MNase preferentially cleaved 140 sites. Clusters of these sites appear to generate the preferential MNase eukaryotic DNA cleavage sites seen on agarose gels at roughly 100 to 300 base-pair intervals. These clusters of preferential cleavage sites rarely occur within gene coding regions. The analysis revealed that duplex DNA sequences preferentially cleaved by MNase are generally determined by a single strand sequence: d(A-T)n, where n greater than or equal to 1, flanked by a 5' dC or dG. Cleavage of the other strand is generally staggered 5' by several nucleotides and occurs even if such sequences are absent on that strand. An empirical predictive DNA cleavage model derived from a statistical analysis of the sequence level data was applied to seven eukaryotic gene loci of known sequence. The predicted patterns were in good general agreement with the previously observed eukaryotic gene/spacer cleavage pattern. Statistical analysis also revealed that sites of predicted preferential DNA cleavage occur less frequently in protein coding regions than for randomized sequences of the same length and nucleotide content. Comparison of the MNase cleavage patterns to the sequence-dependent pattern of binding energies between duplex DNA strands indicates that MNase preferentially cleaves sequences with low helix stability.