Microgravity culture reduces apoptosis and increases the differentiation of a human colorectal carcinoma cell line.

Our hypothesis is that rotation increases apoptosis in standard tissue culture medium at shear stresses of greater than approximately 0.3 dyn/cm2. Human MIP-101 poorly differentiated colorectal carcinoma cells were cultured for 6 d in complete medium in monolayers, on Teflon-coated nonadherent surfaces (static three-dimensional [3D]) or in rotating 3D cultures either in microgravity in low-earth orbit (3D microg) or in unit gravity on the ground (3D 1g). Apoptosis (determined morphologically), proliferation (by MIB1 staining), and the expression of epidermal growth-factor receptor (EGF-R), TGF-alpha, or TGF-beta were assessed by immunohistochemistry, while the expression of the differentiation marker carcinoembryonic antigen (CEA) was assessed on Western blots. Over the course of 6 d, static 3D cultures displayed the highest rates of proliferation and lowest apoptosis. This was associated with high EGF-R, TGF-alpha, and TGF-beta expression which was greater than that of a monolayer culture. Both rotated 3D lg and 3D microg cultures displayed lower expression of EGF-R, TGF-alpha, or TGF-beta and proliferation than that of monolayer or static 3D cultures. However, rotated 3D microg displayed significantly less apoptosis and greater CEA expression than rotated 3D 1g cultures. When rotated cultures of MIP-101 cells were grown uncler static conditions for another 3 d, proliferation increased and apoptosis decreased. Thus, rotation appears to increase apoptosis and decrease proliferation, whereas static 3D cultures in either unit or microgravity have less apoptosis, and reduced rotation in microgravity increases CEA expression.

Classification of small B-cell lymphoid neoplasms using a paraffin section immunohistochemical panel.

Immunophenotypic analysis is critical in categorizing small B-cell neoplasms; however, many recommended antibody panels have required fresh or frozen tissue. Many paraffin-reactive antibodies are now available but have been studied mostly in isolation. Therefore, the utility of a panel of paraffin-reactive antibodies in differentiating small B-cell neoplasms was investigated. Paraffin-embedded sections of small lymphocytic lymphoma/B-chronic lymphocytic leukemia (SLL/B-CLL; 12), mantle cell (MCL; 15), follicular (FL; 11), and marginal zone B-cell (MZL; eight) lymphomas were stained with CD20/L26, CD3, CD43/DF-T1 or Leu22, CD5/4C7, CD23/BU38, cyclin D1/H295, and CD10/56C6 antibodies. For select antibodies, results were compared to flow cytometric data (FC). Formalin and B5 fixation were also compared. Seven of 11 SLL/B-CLL were CD43+ CD5+ CD23+ cyclin D1- CD10-; seven of 11 MCL were CD43+ CD5+ CD23- cyclin D1+ CD10-; nine of 10 FL were CD43- CD5- CD23- cyclin D1- CD10+; and five of six MZL were CD43+ CD5- CD23- cyclin D1- CD10-. CD5, CD23, and CD10 stains showed sensitivities of 81, 88, and 100%, respectively, compared to FC. With B5 fixation, cyclin D1 was more often negative and CD5 more often equivocal. A panel of paraffin-reactive antibodies aids in classification of small B-cell neoplasms, although a small number of cases have indeterminate phenotypes and MZL have no defining features. CD5 separates most SLL/B-CLL and MCL from FL and MZL. CD23 separates SLL/B-CLL from most MCL, but cyclin D1 is most important for identifying MCL. CD10 positivity distinguishes most FL from other small B-cell lymphoid neoplasms.

Hepatocyte growth factor and c-MET in benign and malignant peripheral nerve sheath tumors.

Hepatocyte growth factor (HGF), secreted by mesenchymal cells, has pleiotropic biological activities on several cell types. HGF and its receptor, the c-met proto-oncogene product (c-MET) have been implicated in the genesis and progression of several carcinomas and sarcomas. It has been suggested that MET/HGF autocrine signaling may contribute to tumorigenesis in sarcomas. HGF has been recently found to be a mitogen for rat Schwann cells and to be present in neurofibromas in NF1 patients. In this investigation, we assessed the immunoreactive patterns of HGF and MET in benign and malignant peripheral nerve sheath tumors (PNST) using archival formalin-fixed tissue. The standard avidin-biotin-peroxidase method was used. All benign tumors were negative with HGF. Eight cases of MPNST were positive with both HGF and MET. In some malignant PNST, positivity with both ligand and the receptor may be indicative of an autocrine mediated signal transduction and may implicate HGF/MET in tumor progression. Immunoreactivity with MET was strikingly greater in MPNST in contrast to benign PNST; this finding may prove to be helpful in distinguishing some histologically low-grade MPNST from cellular and atypical benign PNST.

Immunohistochemical characterization of mast cell disease in paraffin sections using tryptase, CD68, myeloperoxidase, lysozyme, and CD20 antibodies.

To date, the diagnosis of mast cell disease (MCD) relied on routine plus histochemical stains. Its differential diagnosis, however, includes a variety of other hematopoietic and particularly B-cell lymphoid neoplasms that are best identified in paraffin sections using immunostains. To determine the paraffin-section immunoreactivity of MCD, 20 specimens from 14 patients with MCD and 1 bone marrow sample (from a patient with probable MCD) that showed equivocal metachromasia, were stained with antitryptase, CD68 (KP-1), CD20 (L26), antilysozyme, and antimyeloperoxidase antibodies. Ten hairy cell leukemias (HCLs), six lymphomas of parafollicular and/or monocytoid B-cell (MBCLs) and low-grade mucosa-associated lymphoid tissue (MALT) types, six granulocytic sarcomas, and five acute myeloid leukemias with monocytic differentiation (M4 and M5 types) were also stained. Tryptase positivity was identified in all of the MCD cases. The staining was moderate to strong in 20 of the 21 specimens, including the probable MCD case. No other neoplasms tested were tryptase positive. CD68 showed similar to even stronger staining in all of the specimens of MCD, HCL, granulocytic sarcoma, and acute myeloid leukemia (M4 and M5 types) tested and in five of the six MBCL and/or MALT-type lymphomas. Weak-to-moderate lysozyme staining seemed to be present in at least 7 of the MCD specimens, whereas there was a lack of staining for myeloperoxidase in 12 specimens, and 7 specimens were nonevaluable (1 case was not tested). Myeloperoxidase was identified in all of the granulocytic sarcomas and acute myeloid leukemias (M4 and M5 types) but not in any HCLs, MBCLs, or low-grade lymphomas of MALT type. CD20 was negative in all of the MCD and myelomonocytic neoplasms but positive in all of the HCLs, MBCLs, and low-grade B-cell lymphomas of MALT type. MCD, therefore, has a characteristic tryptase-positive, CD68-positive, and CD20-negative phenotype in paraffin sections. This distinguishes MCD from the hematopoietic and/or lymphoid disorders that it most closely resembles.

Mixed allogeneic chimerism in lung allograft recipients.

Microchimerism in lung allograft recipients was studied in the autopsies of nine female recipients of male lung grafts who had survived for more than 1 month after transplantation. Using a Y chromosome-specific probe tissues were studied for the presence of donor cells that had migrated beyond the graft itself. They were quantitated by cell counting to give absolute numbers of cells per organ volume. While donor cells were disseminated throughout the body, their numbers were small. These absolute numbers should be studied in a larger group of recipients to determine if they correlate with prognosis and the development of bronchiolitis obliterans.

An immunophenotypic study of the inflammatory cell populations in colon adenomas and carcinomas.

There is increasing interest in the host immunologic response to colon carcinoma as immunotherapeutic techniques are being developed. We studied the inflammatory cells in 27 specimens of normal mucosa, 16 hyperplastic polyps, 21 tubular adenomas, 19 tubulovillous adenomas, 12 villous adenomas, and 17 invasive carcinomas using immunohistochemical techniques in paraffin-embedded tissue. UCHL-1-positive T-cells predominated in the lamina propria of all specimens. In polyps and carcinomas, reactive lymphoid follicles composed of L26-positive B-cells, and UCHL-1-positive T-cells were a prominent feature and UCHL-1-positive cells were increased in the epithelial compartment. Cells bearing surface immunoglobulins were widely distributed in all specimens, with IgA predominating. There was a relative increase in IgG-positive cells in the carcinomas. KP1-positive macrophages, S-100-positive dendritic cells, and HLA-DR-positive cells were oriented toward the lumenal surface in normal mucosa and hyperplastic polyps, suggesting a diffuse antigen presenting system. Macrophages and dendritic cells were increased and dispersed in the neoplasms. HLA-DR expression was increased in the neoplasms, mainly in the stromal cells. We conclude that there is an activated immune response in adenomas and carcinomas of the colon compared to normal mucosa. This is represented by expansion and reorganization of both the T- and B-cell compartments and the macrophage-cell systems.

Multidrug resistance in lung allograft recipients: possible correlation with the development of acute and chronic rejection.

The expression of drug resistance antigens in mononuclear inflammatory cells was studied in transbronchial lung biopsy specimens of lung allograft recipients who experienced steroid-sensitive and steroid-resistant bouts of acute rejection and bronchiolitis obliterans. Immunostains for C494 and C219 epitopes of p-glycoprotein and human metallothionein revealed that (1) mononuclear cells expressing these antigens are present in the lung allograft during rejection, (2) that steroid-resistant acute rejection is associated with increased percentages of C494 and metallothionein-positive cells as compared to steroid-sensitive cases, (3) that bronchiolitis obliterans was associated with a higher percentage of cells with drug-resistant antigen expression, and (4) that steroid-resistant bronchiolitis obliterans is associated with the highest percentage of C494 and metallothionein-positive cells in the five clinical situations studied. P-glycoprotein and metallothionein expression may be a marker of aggressive or persistent cases of acute rejection and bronchiolitis obliterans.

Architectural remodeling of lung allografts in acute and chronic rejection.

The mesenchymal and extracellular matrix alterations that occur in acute and chronic rejection of the lung allograft were studied immunohistochemically, utilizing a wide panel of antibodies. In early rejection, perivascular and peribronchiolar mononuclear infiltrates were associated with basement membrane disruption of the vessels and airways and an ingrowth of muscle-specific actin-, vimentin-positive, desmin-negative spindle cells accompanied by type IV collagenase-positive histiocytes. Subsequent fibrous scarring was manifested by perforation and reduplication of the basement membrane of airways and vessels and dense collagen deposition, primarily type III. As has been suggested in idiopathic pulmonary fibrosis, the fragmentation of basement membranes and the deposition of collagen IV and laminin by mesenchymal cells in vessels and airways may reflect the irreversible fibrosis responsible for allograft dysfunction.

Comparison of extracellular matrix antigens in subtypes of bronchioloalveolar carcinoma and conventional pulmonary adenocarcinoma. An immunohistochemical study.

In contrast to the conventional pulmonary adenocarcinomas (CPAs), bronchioloalveolar carcinoma (BAC) grows predominantly by spreading along the existing alveolar septal framework. Within the BAC category, three subtypes have been identified: mucinous, nonmucinous, and sclerosing BAC. Of these, mucinous and sclerosing BACs have worse prognoses compared with nonmucinous BAC. However, the manifestation of aggressive behavior is different between the mucinous and sclerosing types of BACs. Multifocality is often produced by aerogenous spread, especially in the case of mucinous BACs. To study the differences between the BAC subtypes and the conventional pulmonary adenocarcinomas, we employed a battery of immunohistochemical stains marking the extracellular matrix architecture (laminin, collagen IV, fibronectin, and collagen III), a degradative enzyme against a basement membrane component (anti-type IV collagenase) and cellular receptors for laminin and collagen IV (alpha 2 integrin) on 16 BACs (5 mucinous, 5 nonmucinous, and 6 sclerosing) and 30 CPAs. The mucinous and nonmucinous BACs demonstrated neoplastic epithelial cells growing along a continuous basement membrane. A similar growth pattern with intact basement membrane was noted in the periphery of sclerosing BACs. However, in contrast to mucinous and nonmucinous BACs, all cases of sclerosing BACs showed disruption or complete absence of basement membrane components (laminin and collagen IV) around the embedded glands located centrally in the sclerotic fibrous stroma, as was seen in the basement membrane analysis of conventional adenocarcinomas. Furthermore, increased type IV collagenase activity was seen in the small centrally located embedded glands in comparison to the peripheral glands. These architectural alterations of basement membrane disruption and phenotypic expression of degradative activity may be a reflection of the invasive behavior of the sclerosing BACs and their tendency to produce lymph node metastasis. Although the mucinous BACs did not show evidence of basement membrane disruption, there was a marked increase in their levels of type IV collagenase expression along with consistently low levels of alpha 2 integrin receptor (laminin and collagen IV receptor) expression. These findings may be related to the ability of the mucinous BACs to detach from the underlying basement membrane and spread aerogenously, and is to be contrasted with the stromal infiltration and desmoplasia of sclerosing BACs and CPAs.

Occurrence of human papillomavirus DNA in primary lung neoplasms.

The occurrence of human papillomavirus (HPV) DNA in primary lung carcinomas and in squamous metaplasia of the bronchus was studied using in situ hybridization techniques and commercially available biotinylated DNA probes to HPV subtypes 6/11, 16/18, and 31/33/35. The authors found HPV DNA in six of 20 cases of squamous cell carcinoma and one of six cases of large cell undifferentiated carcinoma. There were two cases each of the 6/11 serotypes and the 16/18 serotypes and three cases of the 31/33/35 serotypes. Infected cells of the squamous carcinomas uniformly showed koilocytosis. No case of adenocarcinoma, bronchioloalveolar carcinoma, or small cell carcinoma was positive (of 32 cases). Areas of squamous metaplasia in infected tumors showed similar HPV DNA expression in 15% of cases, especially in those with condylomatous atypia. In 5.8% of random bronchial biopsies of squamous metaplasia, HPV DNA was identified. The relationship of HPV infection to the development of upper and lower respiratory tract carcinomas is discussed.

Estrogen and progesterone receptors in lymphangioleiomyomatosis, epithelioid hemangioendothelioma, and sclerosing hemangioma of the lung.

The therapeutic options in the treatment of lung neoplasia usually have not included hormonal therapy, unlike those for primary tumors of other sites (e.g., breast). However, two mesenchymal proliferations of lung, lymphangioleiomyomatosis and epithelioid hemangioendothelioma (EHE), and one epithelial tumor, sclerosing hemangioma (SH), have a significant female predilection and may benefit from such hormonal therapy. The authors investigated five cases each of EHE and lymphangioleiomyomatosis and four cases of SH for expression of estrogen and progesterone receptors and 17-beta estradiol in paraffin-embedded tissue. Only one case each of lymphangioleiomyomatosis and EHE expressed 17-beta estradiol. All of the other cases were negative. These findings are contrary to the viewpoint held in published literature, especially in case reports of lymphangioleiomyomatosis, describing patients with positive estrogen and progesterone receptor results. Consequently, a number of issues must be considered in the clinical and immunohistochemical evaluation of the estrogen and progesterone receptor status of these rare pulmonary neoplasms.

Verruca vulgaris of the larynx. Demonstration of human papillomavirus types 6/11 by in situ hybridization.

Verruca vulgaris of the larynx (VVL) is a distinctly uncommon lesion related to the human papillomavirus (HPV). The clinical and pathologic features of a case involving the true vocal cords of a 37-year-old woman are presented and compared with the seven cases previously reported in the English language literature. Papillomavirus capsid antigen was detected in the excised tissue on immunostaining, and viral particles were seen by electron microscopy. In situ hybridization with biotinylated DNA probes clearly demonstrated HPV types 6/11. To our knowledge, this is the first case of VVL in which the virus associated with VVL has been genotyped. The results were unexpected because verruca vulgaris of the skin, lips, and oral cavity is associated with HPV types 2 and 4. This implies that verruca vulgaris can be caused by HPV types other than 2 and 4. In addition, since HPV types 6 and 11 are also the same genotypes associated with multiple papillomatosis of the larynx, it further indicates that VVL is virologically more related to multiple papillomatosis of the larynx than to its counterpart on the skin, lips, and oral cavity. The clinical and pathologic features that distinguish VVL from other similar lesions of the larynx are also discussed.

Use of a biotinylated DNA probe specific for the human Y chromosome in the evaluation of the allograft lung.

A cloned 3.4 kilobase DNA probe derived from the heterochromatin of the Y chromosome was used to investigate the regeneration and reepithelialization of allograft lungs of nine recipients who received sex mismatched donor organs. Patients were monitored for varying periods of time, up to four years, by transbronchial biopsy. In situ hybridization on paraffin-embedded biopsies utilizing the Y probe revealed that bronchial and alveolar epithelium and arterial and venous endothelium of the peripheral lung retained a donor phenotype, irrespective of episodes of acute or chronic rejection (obliterative bronchiolitis) which are known to injure these cellular subsets. In contrast, migratory cells, lymphocytes and macrophages, gradually, at varying rates, infiltrated the allografted lungs, replacing preexisting donor elements. Cases of active OB were manifested by infiltration of bronchioles by sex-mismatched lymphocytes; however, in some instances, quiescent recipient lymphocytes colonized the allograft and were unassociated with histologic rejection. Macrophages of similar sex seemed to cluster together within air spaces. Use of a DNA probe for the Y chromosome and in situ hybridization techniques allow monitoring of cellular alterations over time in recipients with sex mismatched allografts.

Multivisceral intestinal transplantation: surgical pathology.

We report the diagnostic surgical pathology of two children who underwent multivisceral abdominal transplantation and survived for 1 month and 6 months. There is little relevant literature, and diagnostic criteria for the various clinical possibilities are not established; this is made more complicated by the simultaneous occurrence of more than one process. We based our interpretations on conventional histology, augmented with immunohistology, including HLA staining that distinguished graft from host cells in situ. In some instances functional analysis of T cells propagated from the same biopsies was available and was used to corroborate morphological interpretations. A wide spectrum of changes was encountered. Graft-versus-host disease, a prime concern before surgery, was not seen. Rejection was severe in 1 patient, not present in the other, and both had evidence of lymphoproliferative disease, which was related to Epstein-Barr virus. Bacterial translocation through the gut wall was also a feature in both children. This paper documents and illustrates the various diagnostic possibilities.