Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro.

We have studied the physical interaction between macrophages and lymphocytes during the immune response to purified protein derivative of tuberculin (PPD) in vitro. Mixtures of peritoneal macrophages and lymph node lymphocytes from guinea pigs immunized with tubercle bacilli formed cell clusters during 20 h of culture with PPD. The number of clusters produced was correlated to the number of immune lymphocytes in the cultures. Peritoneal macrophages which had been pulsed with PPD and untreated lymph node lymphocytes produced cell clusters in the absence of free PPD in numbers equivalent to those produced by the same cells in the presence of free PPD. In cultures containing a mixture of PPD-pulsed macrophages, not-pulsed macrophages, and immune lymphocytes with no free PPD, cell clusters developed mainly between the antigen-pulsed macrophages and lymphocytes. Cluster formation was antigen-specific with the specificity residing in the lymphocytes, mainly or exclusively in the T lymphocytes. These data indicate that in the process of cell cluster formation macrophages serve as antigen-binding (or -processing) cells, while a subpopulation of lymphocytes interact physically and specifically with the macrophages.

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. II. Ultrastructure of clusters formed during the early response.

Macrophage-lymphocyte clusters are formed when lymph node cells and autologous peritoneal exudate cells from guinea pigs immunized with tubercle bacilli are cultured in the presence of purified protein derivative of tuberculin (PPD) for 20 h. We have studied the ultrastructure of these clusters employing transmission and scanning electron microscopy. The most simple macrophage-lymphocyte cluster consisted of one macrophage, one large central lymphocyte with a blastoid appearance attached to the macrophage with a broad area of contact, and from a few to more than 20 small peripheral lymphocytes attached to the central lymphocyte by their uropods. Some clusters were of more complex type, containing two or three macrophages or one macrophage with more than one central lymphocyte attached to the surface, but even in these clusters each peripheral lymphocyte was attached only to one central lymphocyte. By morphological criteria the peripheral lymphocytes were T lymphocytes.

Cerebral blood flow in rats with renal and spontaneous hypertension: resetting of the lower limit of autoregulation.

The effect of chronic hypertension on cerebral blood flow (CBF) was studied in anaesthetised rats. CBF was measured with the intracarotid 133Xe injection method. Rats with spontaneous and renal hypertension were compared with normotensive controls. The lower limit of autoregulation was determined during controlled haemorrhage. In the normotensive rats, CBF remained constant until mean arterial pressure (MAP) had decreased to the range of 50-69 mm Hg. Thereafter, CBF decreased with each further decrease in MAP. In both types of hypertensive rats, CBF remained constant until MAP had decreased to the range of 70-89 mm Hg. Thus, a 20-mm Hg shift of the lower limit of CBF autoregulation was found in both spontaneous and renal hypertensive rats. A neuropathological study revealed ischaemic brains lesions in half of the hypertensive rats following hypotension, whereas only a single lesion was found in one of six normotensive rats. No ischaemic brain lesions were found in a control study in which CBF was shown to be stable over a 21/2-h period. In conclusion, hypertensive rats showed a shift of the lower limit of CBF autoregulation as well as an increased susceptibility to ischaemic brain damage during hypotension. These findings presumably reflect hypertensive structural changes in the cerebral circulation.

Iliac crest biopsy: representativity for the amount of mineralized bone.

The aim of the study was to evaluate the representativity of iliac crest biopsy for the amount of mineralized trabecular and cortical bone in the skeleton. The following data were obtained on bone from 14 necropsies: right sided iliac crest biopsy, lumbar spine biopsy, dry fat free weight of lumbar spine, bone mineral density (BMD) in the lumbar spine and dry fat free weight of cortical and trabecular bone from the left distal forearm. The amount of mineralized cortical and trabecular bone from various sites was compared by linear regression analysis. The results confirm iliac crest biopsy as a good predictor of the amount of trabecular bone, but not of cortical bone. Furthermore, iliac crest biopsy is a better estimate of the amount of trabecular bone in the lumbar spine than spinal BMD.

Necrotizing vasculitis in athymic rats with infarct kidney hypertension.

Infarct kidney hypertension was induced in congenital athymic nude rats and in their haired normal littermates. In both groups a significant and similar elevation of blood pressure was observed. The mesenteric vessels were studied histologically five, 12 and 20 days after operation. Necrotizing vasculitis with and without perivascular inflammatory reactions was found in mesenteric arteries and arterioles in six out of six athymic and in six out of 11 control rats. In sham operated athymic rats and in haired littermates neither hypertension nor vasculitis was observed. These observations indicate that the thymus play no role in the pathogenesis of acute hypertensive vascular disease.

HLA class I antigens are expressed by Sertoli cells of intratubular germ cell neoplasia.

Five frozen samples from seminomas and one from a combined seminoma/embryonal carcinoma and surrounding tissue were examined for the presence of HLA class I molecules using an antibody directed against a monomorphic epitope of the 45 kDa long chain of HLA-A, -B and -C molecules. Only tubules with intratubular germ cell neoplasia stained positively, while normal seminiferous tubules and invasive tumor cells stained negatively. The immunohistology with anti-HLA class I antibody was compared to that with anti-beta 2-microglobulin antibody which reacts with the light nonpolymorphic chain of all HLA class I molecules. Immunohistology with these two antibodies was indistinguishable. This result combined with immunohistology using anti-PLAP indicates that the stained cells are Sertoli cells. The results are discussed in relation to lymphocytic infiltration and immune surveillance of seminomas.

Sertoli cells, but not tumor cells, of seminoma in situ express ICAM-1.

Tumor and surrounding testicular tissue from six seminomas and one combined seminoma/embryonal carcinoma were examined for the expression of ICAM-1, VCAM-1 and ELAM-1. This was done by immunohistochemical staining of frozen samples using monoclonal antibodies and the avidin-biotin/ peroxidase or alkaline phosphatase staining method. ICAM-1 was expressed by Sertoli cells of intratubular germ cell neoplasia, but not by any of the cells in normal seminiferous epithelium, or by neoplastic germ cells whether invading or not. In addition inflammatory cells and endothelium expressed ICAM-1. VCAM-1, and also occasionally ELAM-1, was expressed only on endothelial cells in and outside the tumors. These results are discussed in relation to lymphocytic infiltration and immune surveillance of seminomas and T-cell tolerance to the antigens of the immunologically privileged seminiferous epithelium.

Fas and Fas-ligand expression in seminomatous testes.

Sixteen seminomas with surrounding tissue containing normal and precancerous (cis) seminiferous tubules were examined for the expression of Fas (CD95, APO-1) and Fas ligand (FasL) (CD95L). This was done by analyzing frozen specimens using immunohistochemistry with antibodies directed against Fas and FasL. The study showed that varying numbers (mean approx. 20%) of Fas-positive lymphocytes were present among tumor-infiltrating lymphocytes, but very few FasL-positive lymphocytes. Fas was not expressed by normal seminiferous tubules and only occasional Fas-positive epithelial cells were seen in cis tubules. FasL was expressed in 9 out of 10 cases in virtually all normal seminiferous tubules, mainly as a thin layer at the base of the seminiferous epithelium. In precancerous tubules, this layer was discontinuous and less pronounced. Rete testis expressed FasL in 2 out of 2 cases with rete present and Fas in 1 out of 1 case. Invasive tumor cells did not express Fas or FasL. The data are discussed in relation to immune reactions to seminomas and to the concept of the testis being an immunologically privileged area.

Sertoli cells of intratubular germ cell neoplasia express beta 2 microglobulin.

The cells in intratubular germ cell neoplasia in the vicinity of 38 germ cell tumors of the testis, including 20 pure seminomas, were studied for the expression of beta 2-microglobulin (beta 2m), the constant component of all HLA class I molecules. Immunohistochemistry using antibodies towards beta 2m, vimentin, placental alkaline phosphatase, and ferritin was employed. Whereas the intratubular cells in normal testis are beta 2m negative, beta 2m positive cells were identified in intratubular germ cell neoplasia tubules in 55 per cent of all tumors and in 60 per cent of the seminomas. The tubules with beta 2m positive cells were located in areas with invasive tumor or in the vicinity of such areas. The beta 2m positive cells were identified as Sertoli cells by morphology and by their staining with anti-vimentin. Neoplastic germ cells, identified by morphology and staining with anti-placental alkaline phosphatase and anti-ferritin were beta 2-microglobulin negative. The most intensely beta 2m-stained Sertoli cells were found in tubules with high concentrations of neoplastic germ cells. Intensely stained Sertoli cells were also found in 'Sertoli cell only' tubules inside invasive tumors and in areas without lymphocytic infiltration. The cells in adjacent normal tubules were beta 2m negative.

Melanotic neuroectodermal tumour as a predominant component of an immature testicular teratoma. Case report with immunohistochemical investigations.

A case of melanotic neuroectodermal tumour (MNT), or so-called retinal anlage tumour, as a predominant component of an immature testicular teratoma is presented. The patient was a 17-year-old man who furthermore had a mature mediastinal teratoma. The MNT was composed mainly of two cell types: small immature neuroblast-like cells and large columnar or cuboidal epithelial-like cells with or without melanin granules. The tumour cells were arranged in solid formations, nests, cords, alveolar and pseudoglandular structures with cleft-like or glomeruloid-like spaces. Myogenic differentiation was found in minor foci. Immunohistochemistry showed both neuroepithelial and mesenchymal features with positive staining reaction for neuron-specific enolase (NSE), S-100 protein (S-100), melanoma antigen (HMB45), cytokeratin and vimentin. Vimentin, desmin and actin were present in the myoid cells. To the best of our knowledge this is the first reported case of MNT originating in the testis. As this tumour component occurred in an immature teratoma, neuroectodermal differentiation of germ cell origin is considered most likely.

Beta 2-microglobulin expression of AIDS-related and classical Kaposi's sarcoma.

The expression of beta 2-microglobulin, the invariable light chain of HLA class I molecules, of Kaposi's sarcoma from 11 AIDS patients and from 11 patients without known immunodeficiency was studied by immunohistochemistry using a polyclonal antibody to beta 2-microglobulin. The staining intensity of spindle cells in these lesions was scored in a semiquantitative system. We found that the spindle cells of Kaposi's sarcomas from AIDS patients showed significantly increased staining intensity for beta 2-microglobulin compared to those of Kaposi's sarcomas from non-AIDS patients. The results may indicate that Kaposi's sarcomas developing in immunocompromised individuals, such as AIDS patients, are not subject to immune selection by T cells eliminating HLA class I high-expressing tumor cells, while this may be the case in non-AIDS patients. Alternatively, the results may be caused by differences in the activity of cytokines, which upregulate the expression of HLA class I molecules on the cell surface.

Expression of beta 2-microglobulin by premalignant epithelium.

Many human tumors express low amounts of HLA class I molecules relative to the normal cells from which they are derived. From experimental work it is clear that the malignant behavior of a tumor cell may depend on its MHC class I expression. Therefore, it is of obvious interest to study the HLA class I expression of human tumors in their various stages. We have studied the HLA class I expression by the cells in premalignant epithelial lesions and invasive carcinoma of the bladder and uterine cervix using immunoperoxidase staining for beta 2-microglobulin of paraffin-embedded tissue. We here assume that beta 2-microglobulin expression by malignant and premalignant cells equals HLA class I expression. Thirty-two of the 36 invasive tumors expressed less overall beta 2-microglobulin than cells from the normal epithelium. In contrast, approximately two-thirds of 34 premalignant bladder epithelia and 47 premalignant cervix epithelia displayed higher overall beta 2-microglobulin expression than the normal epithelium. Thus, a systematic large-scale elimination of HLA class I high-expressing tumor cell variants may take place only after the tumor penetrates the basement membrane.

Are pathologists biased by clinical information?: A blinded cross-over study of the histopathological diagnosis of mesothelial tumours versus pulmonary adenocarcinoma.

In a blinded cross-over design, we studied whether three pathologists were biased by clinical information when making histopathological diagnoses of adenocarcinoma of the lung and benign and malignant mesothelial tumours. Furthermore, the interobserver variation of these diagnoses was assessed. Forty-one cases of adenocarcinoma of the lung and mesothelial tumours were assessed by three pathologists in four rounds. In the first two rounds, slides stained by H&E and clinical information were available. Slides and information were matched so that a specific slide in one round was given clinical information suggesting adenocarcinoma and in the other round, the clinical information suggested mesothelial tumour. In the third and fourth rounds, a panel of immunohistochemical stains was added. The clinical information was matched in the same way as in the first and second rounds. Bias by clinical information was observed when the diagnoses were made on slides stained by H&E, while no bias could be demonstrated when immunohistochemical reactions were included. The reproducibility also improved significantly when these slides were available.

Nitric oxide synthase expression of oligodendrogliomas.

In the central nervous system, nitric oxide (NO) has a variety of biological functions including vasorelaxation and neurotransmission. The synthesis of NO is catalyzed by NO synthases (NOS) existing in 3 isoforms, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). NO synthase has implications in the pathophysiology of primary glial brain tumors with enhanced expression of nNOS and eNOS in high-grade astrocytic tumors, WHO grades III and IV. Only minor groups of pure oligodendrogliomas have been investigated. The aim of the investigation was to study the expression of the 3 NOS isoforms in this genetically divergent group of primary gliomas and to correlate the findings with tumor grade and expression pattern for the major group of gliomas--the astrocytomas. We examined the NOS expression in 35 oligodendrogliomas, WHO grade II, and 7 anaplastic oligodendrogliomas, WHO grade III, by immunohistochemical methods using formalin-fixed paraffin-embedded material. We observed only a minor expression of nNOS and sparse expression of eNOS in the tumor cells, but a vivid expression of eNOS in the vascular endothelial cells in both the tumor and the surrounding tissue. The rich expression of eNOS in oligodendroglioma vessels independent of tumor grade may suggest that blood flow and angiogenesis in these richly vascularized tumors are modified by NO. Interestingly, enhanced expression of inducible NOS was observed in the oligodendroglial tumor cells in 19 of 35 oligodendrogliomas (54%) and in 2 of 7 anaplastic oligodendrogliomas (29%). This is diverging for iNOS expression in astroglial tumors and the data could be indicative of iNOS exerting anti-tumor activity which may protract the progression from low-grade oligodendrogliomas to more anaplastic types.

Immunohistochemical investigation of p53 and EGFR expression of oligodendrogliomas.

The biological behavior of oligodendrogliomas is somewhat unpredictable. A supplementing prognostic factor is, therefore, desirable. Thirty-two supratentorial pure oligodendrogliomas were studied immunohistochemically by exposing the tumors to a monoclonal antibody towards the p53 protein, and a polyclonal antibody against the epidermal growth factor receptor (EGFR). A mean p53 labeling index (% of tumor cells stained) of 8.6% and a weak EGFR expression in 18 of the oligodendrogliomas were found. Univariate analysis showed no correlation between p53, EGFR expression and prognosis. Multivariate analysis showed that age was a prognostic factor for survival.

Nitric oxide synthase expression and enzymatic activity in human brain tumors.

Nitric oxide (NO) is synthesized by NO synthases (NOS), existing in 3 isoforms. NO influences a great variety of vital functions including vascular tone and neurotransmission. Under conditions of excessive formation, NO emerges as an important mediator of neurotoxicity in a variety of disorders of the central nervous system (CNS). Inhibitors of NOS are available that may modify the activity of all isoforms, which may be of clinical relevance. The expression of the 3 NOS isoforms nNOS, iNOS and eNOS and NOS enzymatic activity was examined in 40 patients with primary CNS tumors (gliomas WHO grades I - IV and meningeomas WHO grades I - III) and in 13 patients with metastases from adenocarcinomas or malignant melanomas. A polyclonal antibody directed against nNOS and monoclonal antibodies directed against iNOS and eNOS were used for immunohistochemical staining. NOS enzymatic activity, measured by labeled arginine to citrulline conversion, was assessed in tissue specimens obtained from the same tumors. NOS data were compared with clinical variables and the degree of edema as judged from MR scanning. nNOS expression was increased in tumor cells of glial neoplasms and most pronounced in high-grade tumors, WHO grades III and IV, and in the carcinoma and melanoma metastases. Low-grade gliomas, WHO grades I and II and meningeomas expressed no or only little nNOS. iNOS was only expressed in a few tumors. eNOS was expressed sporadically in the tumor cells while the expression was increased in vascular endothelial cells in both the tumor itself and the peritumoral area of glial neoplasms, and in metastases. eNOS expression was sporadic in endothelial cells of meningeomas. NOS enzymatic activities were heterogeneous among tumor types (0 - 13.8 pmol/min/mg of protein) without correlation to the NOS expression found by immunohistochemical techniques. Likewise, NOS activity and expression was not correlated to the clinical scores or brain edema. In conclusion, nNOS expression may be a putative useful indicator of brain tumor differentiation and malignancy. The enhanced expression of eNOS in vascular endothelial cells of glial neoplasms and metastases raises the possibility that NO production in tumor endothelial cells may contribute to tumor blood flow regulation and possibly brain edema.

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. IX. Antigen-pulsed macrophages as a tool for specific absorption of cluster-initiating T cells.

T-cell populations from guinea-pigs sensitized to the protein antigens purified protein derivative of Mycobacterium tuberculosis, ovalbumin, or horseradish perioxidase can be selectively depleted of cells capable of initiating antigen-specific macrophage-lymphocyte clusters in vitro. The depletion is achieved by incubating the T cells on a monolayer of antigen-pulsed macrophages in a Petri dish for some hours and then gently aspirating the cells not adhering to the bottom of the dish. When subsequently assayed, the aspirated cells were found to be depleted of cluster-initiating lymphocytes committed to horseradish peroxidase, monolayers of macrophages pulsed with that antigen must be used. The optimum time for incubation on the absorbing monolayer appears to be 4 h, and two successive incubations are more effective than one. The cell density of the absorbing monolayer and the handling of the Petri dish may be critical for effective removal of the cluster-initating lymphocytes. With optimum procedure we have achieved up to 90% depletion of specific cells with no depletion of cells committed to a control antigen.

Malignant non-Hodgkin lymphoma of the gastrointestinal tract. A histopathological review of 34 cases.

Thirty-four cases of malignant non-Hodgkin lymphomas of gastro-intestinal origin were histopathologically reviewed employing Rapaport's (18) classification. The majority of the tumours, 25, were localized to the stomach. The rest of the lesions occurred with decreasing frequency throughout the gastro-intestinal tract, with the exception of 3 rectal cases. Thirty lymphomas displayed a purely diffuse pattern, 1 was purely nodular, and 3 were nodular with diffuse components. "Histiocytic" lymphoma was the commonest type, including 16 cases; poorly differentiated lymphocytic type next, with 10 cases. The histopathology of this series does not differ from those of comparable studies.

No prediction of recurrence of meningiomas by PCNA and Ki-67 immunohistochemistry.

Immunohistochemistry for the expression of the proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 was studied in 16 non recurring meningiomas, 11 meningiomas recurring as benign tumors, 6 recurring as atypical meningiomas and in 9 recurring as malignant meningiomas. Non recurring meningiomas were defined in this study as tumors without recurrence at least 8 years after surgery. In addition 16 benign recurrences, 14 atypical- and 12 malignant meningiomas were studied. In each group great variation of labeling indices (LI) = per cent of tumor cells labeled was observed, especially of PCNA LIs. The non recurring meningiomas displayed lower mean LI for PCNA and Ki-67 than did the recurring meningiomas of all groups but the differences were not statistically significant. The same pattern was seen when totally resected tumors were studied alone. Benign-, atypical-, and malignant meningiomas had labeling indices that were related to the grade of malignancy. Only PCNA LIs of atypical- and malignant meningiomas were statistically significantly higher than PCNA LIs of non recurring meningiomas. The study indicates that PCNA and Ki-67 are of minor value as predictors of recurrence of benign meningiomas.