Transplantable myeloproliferative disease induced in mice by an interleukin 6 retrovirus.

Lethally irradiated mice transplanted with bone marrow cells infected with a novel recombinant retrovirus (murine stem cell virus-interleukin 6 [MSCV-IL-6]) bearing a mouse IL-6 gene developed a fatal myeloproliferative disease within 4 wk of engraftment. The hematologic manifestations of the syndrome included elevated peripheral leukocyte counts (up to 430 x 10(3) cells/mm3) with a predominance of neutrophilic granulocytes, microcytic anemia, and thrombocytosis or thrombocytopenia. The mice showed extensive neutrophil infiltration of the lungs, liver, and occasionally lymph nodes, plus splenomegaly resulting from enhanced splenic myelopoiesis (30-60-fold increase in progenitor numbers). Despite the chronic stimulation of neutrophil excess by IL-6, bone marrow from affected mice was capable of repopulating the hematopoietic tissues (bone marrow and spleen) of lethally irradiated hosts during repeated serial transplantation. In the longest documented case, the progeny of a single MSCV-IL-6-marked cell transferred the myeloproliferative disease to two secondary, four tertiary, and two quaternary recipients (the clone endured for a total of 72 wk). These results, demonstrating considerable proliferative longevity of the IL-6-producing cells, support an in vivo role of IL-6 in the maintenance of hematopoietic precursors. Dysregulated IL-6 production also had significant systemic effects. The mice displayed increased mesangial cell proliferation in the kidney, frequent liver abnormalities, and marked alterations in plasma protein levels. Unlike previous studies where constitutive expression of exogenous IL-6 genes resulted in lymphoproliferative disorders characterized by massive plasmacytosis, minimal plasma cell expansion occurred in the MSCV-IL-6 mice during the observation period. Potential explanations for the differences in disease phenotypes observed in the present and previous studies are different cell types expressing the exogenous IL-6 genes, higher sustained circulating levels of IL-6 achieved using the MSCV-IL-6 retroviral delivery system, and/or the premature death (3-15 wk after transplantation) of the MSCV-IL-6 mice before the onset of plasmacytosis. This animal model should prove useful for further investigation of the function of IL-6 in normal and abnormal hematopoiesis and in inflammatory responses.

Blastomycosis in a young African man presenting with a pleural effusion.

Blastomyces dermatitidis is a dimorphic fungus endemic to north-western Ontario, Manitoba and some parts of the United States. The fungus is also endemic to parts of Africa. Pulmonary and extrapulmonary findings of a 24-year-old African man who presented with weight loss, dry cough and chronic pneumonia not resolving with antibiotic treatment are presented. The unusual occurrence of pulmonary blastomycosis associated with skin lesions and a moderate pleural effusion is reported.

Treatment of advanced Hodgkin's disease with chemotherapy--comparison of MOPP/ABV hybrid regimen with alternating courses of MOPP and ABVD: a report from the National Cancer Institute of Canada clinical trials group.

PURPOSE: This randomized, prospective trial compares outcomes for patients with advanced Hodgkin's disease treated with mechlorethamine, vincristine, procarbazine, and prednisone (MOPP)/doxorubicin, bleomycin, and vinblastine (ABV) hybrid regimen or alternating MOPP/doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD). METHODS: Three hundred one patients with advanced Hodgkin's disease were randomized to receive MOPP/ ABV hybrid regimen or alternating MOPP/ABVD after stratification for prior treatment, B symptoms, and treatment center. Eligible patients were either previously untreated and found to have stage IIIB, IVA, or IVB disease or previously treated with wide-field irradiation. Responding patients received a minimum of eight cycles of chemotherapy. Those with residual disease in a localized region received irradiation between the sixth and seventh cycle of treatment. RESULTS: Response rates to the two regimens were similar. Five-year overall survival rates were 81% and 83% for MOPP/ABV hybrid and alternating MOPP/ ABVD, respectively (P = .74; 95% confidence interval [CI] for the difference, -11% to 7%). Five-year failure-free survivals were 71% and 67% for MOPP/ABV hybrid and alternating MOPP/ABVD, respectively (P = .87; 95% CI for the difference, -9% to 17%). Significantly more episodes of febrile neutropenia and stomatitis were observed with the MOPP/ABV hybrid regimen; there was no significant difference in fatal toxicity. Patients with predefined, high-quality partial responses (PR-1s) had results similar to those with complete responses (CRs). Planned subset analysis showed no significant difference in outcome between the two arms of the trial for patients with newly diagnosed disease (5-year failure-free survival rates were 70% for MOPP/ABV hybrid and 59% for alternating MOPP/ABVD; P = .180), but superiority of alternating MOPP/ABVD for patients with prior irradiation (5-year failure-free survival 94% v 73%; P = .017). CONCLUSION: MOPP/ABV hybrid and alternating MOPP/ABVD regimens are equally effective for patients with advanced Hodgkin's disease.

The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue.

Encouraged by recent reports of extraction of diagnostically useful DNA from formalin-fixed tissues, we devised a study to determine which fixatives are suitable for DNA hybridization studies. Seven lymph node specimens (6 lymphomas and one reactive hyperplasia) were studied. Specimens were divided into portions, each of which was processed separately. Processing protocols followed were: 1) snap freezing; 2) immersion in formalin, ethanol, glutaraldehyde, B5 and methanol acetic acid (MAA) Michel's transport medium or phosphate buffered saline for 2, 24, 48 and 120 hrs.; 3) paraffin embedding following fixation. DNA was obtained by proteinase K digestion followed by phenolchloroform extraction. DNA was cleaved with restriction endonucleases (Eco RI and Bam HI), separated by agarose gel electrophoresis, after which Southern blot hybridization with radio-labelled probes for J-heavy and T-beta genes was performed. Fixation with formalin, glutaraldehyde and B5 resulted in poor yields of DNA. MAA produced degradation of DNA and Michel's medium and PBS gave low quantities and purity of extracted DNA. Ethanol fixation consistently permitted extraction of large amounts of high molecular weight DNA suitable for hybridization studies and compared favourably with the fresh-frozen 'gold standard'. We conclude that ethanol may be the fixative of choice when transport or storage conditions limit the availability of fresh frozen tissue for DNA hybridization studies.

Creating low-power photomicrographs using a 35 mm digital slide scanner.

A method for obtaining high quality photomicrographs at very low power magnification using a digital slide scanner is described. Using an easily modified 35-mm film slide mount, it is possible to scan a conventional 25-mm wide glass slide and to obtain an image that is uniformly in focus and of even illumination. The computer bitmap file that is generated is suitable for computer presentations or publication-quality prints and may be magnified up to 12-fold without significant image degradation.

The varied histopathology of lymphadenopathy in the homosexual male.

We review the clinical features and histopathologic changes observed in 69 cases of lymphadenopathy in homosexual men. The most common pattern seen was that of florid reactive follicular hyperplasia (43 cases). A peculiar and distinctive lysis of the germinal centers, a phenomenon we have termed "follicle lysis," was noted in 25 cases. Eighteen of these lymph nodes also contained sinusal collections of "monocytoid" cells and neutrophils and six showed focal dermatopathic changes. Seven cases were characterized by a lymphocyte-depleted pattern with only occasional regressively transformed germinal centers. Nine patients were found to have involvement of their lymph nodes by Kaposi's sarcoma; malignant lymphomas were encountered in 10 patients (eight with Hodgkin's disease and two with non-Hodgkin's lymphomas). Polykaryocytes (multinucleated giant cells) were observed in germinal centers or interfollicular areas within the lymph nodes of four patients. Limited clinical follow-up was available but the lymphocyte-depleted group appeared to have a more aggressive clinical course (three patients in this group developed pneumocystis pneumonia, two had mycobacterial infections and one cutaneous Kaposi's sarcoma). The diverse nature of these findings and the potential for treatment of certain patients (i.e., those with malignant lymphomas and mycobacterial infections) underlines the importance of lymph node biopsy in all cases of unexplained lymphadenopathy in homosexuals and other individuals susceptible to the acquired immunodeficiency syndrome (AIDS). The histopathologic findings may also serve to identify a subgroup of these patients at increased risk to develop more severe AIDS-related complications.

Langerhans' cell granulomatosis (histiocytosis X) associated with malignant lymphomas.

We present six patients in whom Langerhans' cell granulomatosis (histiocytosis X) was found in lymph nodes also harboring malignant lymphomas: Hodgkin's disease in five and non-Hodgkin's lymphoma in one. This brings to 12 the total number of such reported cases. Whether this represents a chance association of the two processes or a peculiar reaction of Langerhans' cells to the lymphoma is unknown. The focal intimate intermingling of the two processes and the inability to identify LCG as an incidential finding in other cancers suggests that this phenomenon may represent a peculiar reaction to the lymphoma.

Differential diagnostic features of nodular L & H Hodgkin's disease, including progressive transformation of germinal centers.

The histologic features in 171 cases of nodular lymphocyte-predominant (L&H) Hodgkin's disease are presented and the association with an abnormal form of follicular hyperplasia termed "progressively transformed germinal centers" (PTGC) by Lennert is discussed. PTGC may closely resemble the nodules of L&H Hodgkin's disease and in 18% of our cases the two processes coexisted in the same lymph node. In addition, two patients had lymph node biopsies showing PTGC prior to biopsies showing nodular L&H Hodgkin's disease and three patients with histologically proved Hodgkin's disease were found to have PTGC in subsequent lymph node biopsies. Immunologic studies on frozen tissue sections from three cases of nodular L&H Hodgkin's disease showed that the neoplastic nodules contained abundant dendritic reticulum cells and B-lymphocytes with scattered T-lymphocytes. These findings suggest that the association between PTGC and nodular L&H Hodgkin's disease is more than coincidental and that this form of Hodgkin's disease preferentially involves B-cell areas of the lymph node, in contrast to the T-zone distribution in other forms of this disorder.

Mutations in the hemagglutinin and matrix genes of a virulent influenza virus variant, A/FM/1/47-MA, control different stages in pathogenesis.

The mouse adapted strain of influenza A/FM/1/47 virus, FM-MA, has increased virulence due to mutations in HA, M1 and at least one other, unmapped, genome segment. Genetic reassortants that differ due to the HA or M1 mutations were used to define the role of these mutations in pathogenesis. Pathological changes in lungs of infected mice were assessed by hematoxylin phloxine saffron (HPS) staining, and viral infection was measured by fluorescent antibody staining of thin sections and flow cytometry of lung parenchymal cells. HA played a role in bronchiolar pathology by increasing necrosis of bronchiolar epithelium, peribronchiolar lymphocytes, and airway obstruction. The HA mutation was shown to be responsible for a 0.2 unit decreased in the pH optimum of fusion and controlled resistance to alpha and beta inhibitors of hemagglutination. Both these changes in biology may confer a replicative advantage in bronchioles seen in the first day of infection. Thus the HA mutation may have conferred a survival advantage in the extracellular lung environment. The M1 mutation resulted in improved growth in the lung and cultured cells and was associated with increases in recruitment of macrophages, spread of infection into the alveoli of the lung and interstitial pneumonia. Sequence analysis indicated that the unmapped mutation in the control of FM-MA virulence is either the K482-->R substitution in the PB2 protein or the D538-->G substitution in the PB1 protein. One or other of these mutations results in a growth advantage in infected lung but not in cultured cells as well as a further increased recruitment and infection of macrophages in the lung. Infection with virulent strains of influenza that induced increases in macrophage recruitment caused hypothermia in the mouse.

In vivo induction of tumor variants by phorbol 12-myristate 13-acetate.

Malignant cells within solid tumors commonly exhibit phenotypic changes such as alterations in karyotype and acquisition of the ability to invade and to metastasize. We have used a fibrosarcoma, grown subcutaneously in C57BL/6 mice, to study the mechanisms underlying this phenotypic instability. Tumor-bearing animals were injected with phorbol myristate acetate (PMA) and then the tumors were transplanted to animals without further PMA treatment. These tumor cells were tum+, that is, unlike the parental tumors, they were able to grow in animals immunized against the parental tumors. This property was maintained for at least 6 tumor passages after the initial PMA injections. Thus, PMA appears to be able to induce an unstable tum+ phenotype in these cells at relatively high frequency.

Lymphocyte predominant Hodgkin's disease: clinical presentation and results of treatment.

The records of 59 patients with lymphocyte predominant Hodgkin's disease (LPHD) evaluated and treated at Stanford University Medical Center between 1963 and 1983 were reviewed. Of these 59 patients, 92% are alive at 10 years following treatment, 78% are relapse-free, and none have died of Hodgkin's disease. Compared with the other histologic subtypes of Hodgkin's disease, LPHD presents more frequently as stage I or II disease (78% vs. 55%) and less frequently with constitutional symptoms (7% vs. 32%). Despite these factors, there is no statistically significant difference in either survival or freedom-from-relapse (FFR) between the histologic subtypes when comparisons are made on a stage-for-stage basis. Analysis of sites of presentation and relapse reveals that LPHD rarely involves intrathoracic structures. Patients with C.S. I disease presenting in inguinofemoral or high cervical lymph nodes do not require staging laparotomy as none of these patients were upstaged by surgery. Patients with stage I disease involving high cervical lymph nodes may be treated with limited field irradiation employing fields no more extensive than a mantle and Waldeyer's ring field, as no relapses have been seen in such patients treated in this fashion. Although limited field irradiation was used successfully for LPHD presenting in other localized sites, inadequate patient numbers preclude assessment of this treatment for those clinical presentations.

Leukocytosis in mice following long-term reconstitution with genetically-modified bone marrow cells constitutively expressing interleukin 1 alpha or interleukin 6.

Leukemic cells of patients with acute myeloid leukemia have recently been shown to spontaneously produce autostimulatory IL-1 and IL-6. In order to investigate the effects of systemic production of these cytokines on normal hematopoietic cells, mice were engrafted with bone marrow cells infected with high-titer retroviral vectors carrying the murine IL-1 alpha or IL-6 genes and the neomycin phosphotransferase gene. Sustained expression of the introduced IL-1 alpha and IL-6 genes was documented by Northern-blot analysis of RNA from G418-resistant mast cells and T cells, derived from bone marrow and spleen, respectively, of successfully reconstituted mice 6-10 months after transplantation. A single mouse engrafted with IL-1 alpha-infected cells which presented with a dramatic neutrophilic granulocytosis (54-fold elevation in circulating neutrophils) was sacrificed for health concerns 2 months post-transplant. Modest changes in peripheral leukocyte counts (at most a 2-fold rise) were observed in all of the other mice, and they remained healthy throughout the observation period. The majority displayed increased hematopoietic activity in bone marrow and spleen, predominantly granulopoiesis, with moderate lymphoid hyperplasia seen in the spleens of mice constitutively expressing IL-1 alpha. These mouse models provide the opportunity to evaluate the potential of persistent IL-1 alpha and IL-6 expression to contribute to leukemogenic transformation.

A case of a Ki-1 large cell anaplastic lymphoma with ultrastructural features.

A 29-year-old woman first presented in 1982 with a large cell lymphoma, initially thought to be of histiocytic origin on the basis of prominent sinus infiltration. She had a complete response to chemotherapy, but relapsed in 1987. Histologically, a repeat biopsy was identical to the first. Immunocytochemically, there was strong reactivity with Ki-1 antibody and histiocyte lineage markers, but all specific T cell markers were negative. Southern blot hybridization demonstrated a clonally rearranged band with the T cell receptor (T beta) probe. Ultrastructurally, the cells showed sparse organelles except for prominent paranuclear Golgi apparatus, frequent reniform nuclear indentations, and ruffled cytoplasmic membranes. This case appears to represent a Ki-1-positive lymphoma with the hybrid features of an activated T lymphocyte and a histiocyte.

Fatal post-transfusion acquired immunodeficiency in a heterosexual man: quantitative lymph node immunopathology.

Employing a novel panel of monoclonal antibodies, we characterized in detail the quantitative immunopathologic alterations of lymph nodes in a 69-year-old heterosexual man who died of acquired immunodeficiency 1.5 years after transfusion of blood products. Absolute decreases were evident in total T cell, helper T cell, cytotoxic T cell, and Leu 8+ cell counts. Suppressor T cells and histiocytes were absolutely increased. There were relative increases in B cells and Leu 7+ cells. B-cell follicles were sparse and exhibited small, burned-out germinal centers composed predominantly of R4/23+ dendritic reticulum cells. These findings closely paralleled those described previously for homosexual men with acquired immunodeficiency syndrome (AIDS). They suggest that AIDS occurring among diverse clinical groups, such as homosexual men and blood donor--recipient pairs, involves similar immunopathologic alterations within lymph node microenvironments.

Pattern of distribution of intraductal and infiltrating ductal carcinoma: a three-dimensional study using serial coronal giant sections of the breast.

The purpose of this study was to establish the 3-dimensional (3D) structure of the breast tissue and to study the distribution and relationship between the intraductal and infiltrating components of ductal carcinoma and other proliferative epithelial lesions of the breast. Thirty mastectomy specimens with infiltrating carcinoma less than 3.0 cm in diameter were serially cut in the coronal plane. Each giant section was divided into small sections for routine processing. Using Photoshop (Adobe) and PowerPoint (Microsoft) software programs, the routinely stained sections were scanned and assembled to reestablish complete giant sections of the breast and subsequently the 3D structure. Intraductal and infiltrating ductal carcinomas, epithelial hyperplasia with atypia, and marked epithelial hyperplasia without atypia were mostly confined to a single duct (27 cases), resulting in an increase in size of the involved breast segment. Three remaining cases included a case of Paget's disease with tumor appearing to spread from one duct system to another system through the epidermis and two cases with multiple separate foci of carcinomas located in different quadrants and accompanied by ductal spread in different lactiferous ducts. Both intraductal and infiltrating carcinomas were often located in the superficial segments (near the subcutaneous tissue) (28 cases). The infiltrating components were often located adjacent to area of pure intraductal carcinoma and were often peripheral (nearer the chest wall than the nipple). Intraductal carcinomas showed a "fanned out" pattern of distribution, frequently extended toward the nipple (with involvement of the nipple or subareolar tissue in 7 cases), and occasionally were seen in the breast tissue peripheral to the infiltrating carcinoma. Multiple ducts with intraductal carcinoma could be seen to be connected with each other with serial sections. However, in at least 6 cases, foci of intraductal carcinomas were separated from each other by segments of duct with benign epithelium. Breast carcinoma often arise from the breast segment close to the subcutaneous tissue. Infiltrating carcinoma lesser than 3.0 cm in diameter is usually located adjacent to the area of pure intraductal. The pattern of spread of intraductal carcinoma has a pyramid-like shape, with the summit toward and occasionally extending up to the nipple. These findings should be considered in the surgical strategy for segmental resections of breast carcinomas.

Ki-1-positive non-Hodgkin's lymphomas. An immunophenotypic, ultrastructural, and morphometric study.

The authors studied four cases of diffuse, large cell non-Hodgkin's lymphomas with strong Ki-1 expression to determine if they shared characteristic ultrastructural or morphometric features in common. The tumor cells in these lymphomas all had very abundant cytoplasm, which often showed a concentration of organelles, especially mitochondria and Golgi apparatus, in regions next to nuclear concavities. One case had very complex cytoplasmic membrane projections resembling a "microvillous" lymphoma. These ultrastructural features combined with the frequent reniform nuclei and scattered lysosomes suggested histiocyte differentiation. However the immunophenotype was variable, more often showing "activation" markers and abnormal T-cell marker patterns. The mean nuclear area for all cases was between 38.3 +/- 12.7 to 42.3 +/- 16.2 microns2, roughly twice the size of the small lymphocyte population. The nuclear contour index, a measure of irregularity of the nuclear membrane, ranged from 4.75 +/- 0.86 to 6.36 +/- 1.89 (a circle is 3.54). These nuclear parameters are comparable in size to those of previously measured large cell lymphomas but exhibit more nuclear irregularity than most. Ki-1 lymphomas appear to represent a somewhat diverse group of tumors of lymphoid origin that exhibit some hybrid features of histiocytes.

Histiocytosis X of the thymus in association with myasthenia gravis.

A patient with myasthenia gravis underwent thymectomy following the failure of medical treatment. The thymus was normal except for four nodules, which, on histologic and electron microscopic examination, were found to consist of proliferative clusters of Langerhans' cells characteristic of histiocytosis X. This patient therefore had two disease processes in which thymic abnormalities are frequently encountered, but their simultaneous occurrence in the same patient has not hitherto been documented. The case is briefly discussed in the context of current concepts of the pathogenesis of histiocytosis X and myasthenia gravis.

CD30 positive (Ki-1) large cell lymphoma presenting with pericardial constriction.

It is very uncommon for the initial presentation of malignant lymphoma to be one of cardiac involvement and for such involvement to precipitate cardiac dysfunction attributable to constriction. We describe a CD30 positive (Ki-1) anaplastic large cell lymphoma, T-cell type, in a 29-year-old man whose presentation was a short history of profound hemodynamic impairment and whose clinical course was rapidly fatal. This patient's constrictive physiology was attributable to diffuse infiltration of the pericardium and epicardium by the Ki-1 lymphoma. Our description of this patient is noteworthy, given that the clinical and pathologic features of Ki-1 lymphomas are still being characterized.

Hemophagocytic syndrome complicating cardiac transplantation.

Infection-associated hemophagocytic syndrome is one of the hemophagocytic disorders, and is most often seen in the pediatric population, typically in the setting of immunosuppression. We present the case of a 33-year-old man who had been well for more than 3 years following cardiac transplantation until he developed the infection-associated hemophagocytic syndrome. The patient had a fulminant downhill course, dying in shock 10 weeks after his first presentation. Serologic studies for Epstein-Barr virus suggested a remote infection; other viral and microbiologic studies were negative. The only previous report of infection-associated hemophagocytic syndrome complicating cardiac transplant appears to be that of a pediatric patient. The case presented illustrates the difficulties in antemortem diagnosis of this disorder, and in its treatment.

Molecular genetic markers in lymphoproliferative disorders.

The elucidation of the genetic mechanisms involved in the generation of antibody diversity is little more than a decade old, but the impact has already been felt in many areas of biology and medicine. In order for a B or T cell to become a functional antigen recognizing cell it must produce a unique protein to act as a membrane receptor. In the case of the B cell this is an immunoglobulin molecule while in the T cell it is an analogous dimeric protein, the T cell receptor. Diversity in these proteins is generated by a shuffling of the genes responsible for these respective proteins and is unique to each cell. Using Southern blot hybridization to exploit this, it is possible to determine whether a group of lymphocytes is derived from a single progenitor cell, i.e., clonal, and also whether they are of B or T cell origin. Oncogene activity and chromosomal translocations are believed to be responsible for a variety of lymphomas and leukemias. The association of established oncogenes, such as c-myc, with translocations to regions near immunoglobulin enhancers in some lymphomas has prompted successful searches for novel proto-oncogenes in other forms of lymphomas that also have characteristic, but different, translocations.