Cellular immune response in atypical tuberculosis diagnosed by PCR in paraffin embedded material.

The aim was to evaluate the cellular immune response in atypical tuberculosis and granulomatous inflammation consistent with tuberculosis (TBC), negative histochemically for acid-fast bacilli and analysed by PCR for Mycobacterium tuberculosis (MT) detection in paraffin-embedded tissue. Thirty six samples of differently localized atypical tuberculous lesions and granulomatous tuberculoid lesions negative for acid fast bacilli and 4 positive cases on Ziehl-Nielsen stain were analysed by PCR for MT detection and were tested immunohistochemically (IHC) for the cellular immune response in the granulomas and perigranulomatous tissue. The samples selected were: 7 pulmonary and 33 extrapulmonary specimens, especially lymph nodes. Histologically, the atypical tuberculous lesions contained supurative necrosis, defective granulomas and cellular polymorphism. The epithelioid cells showed frequent mitoses. The immunoprofile of cells was polymorphous. L26 positive small lymphocytes were found in nodular lymphoid aggregates surrounding granulomas. A significantly increased number of positive UCHL1 cells were found in 33 out of the 40 analysed cases, with a larger percentage of CD4 positive T cells (81.8% of cases). CD44 was positive in multinucleated giant cells (17.5% of cases), epithelioid cells (60% of cases) and lymphocytes (30% of cases). CD68 was localized in multinucleated giant cells and epithelioid cells, in a 4%, respectively 62.5% of cases. The PCR was performed in all 40 cases; the tissue samples were heterogeneous (lung, lymph nodes, lever, nasopharynx, etc.) and needed a good quality extraction of DNA. Performing a control PCR for Beta Globin tested the extraction; a good result was obtained in 31 cases (77.5%); from these, 19 cases had amplification for IS 6110. The cellular immune response in the atypical tuberculous lesions was similar in cases with and without acid-fast bacilli, but positive for PCR. In the most cases with negative PCR reaction, it was due to a deficient fixation of the material. The T lymphocytes were numerous in all types of tuberculous granulomas, with the prominence of CD4 positive subtype. The immunoprofile of the epithelioid cells, positive for CD44 and CD68, presenting frequently mitoses suggests an activate state in a possible relationship to the T-cell-mediated immune response in tuberculosis.

Electronmicroscopic and histoenzymological researches regarding myocardial biology in some malformations.

A number of 65 heart malformations, taken from intraoperatory biopsies at different degrees of congestive heart failure were histological, histoenzymological and electronmicroscopical analyzed. It was followed the pathogenesis of the lesions determined by the hemodynamic and hypoxic intracardiac disorders. These hadn't specificity and were disseminated in different evaluative degrees, being predominant in certain myocardial areas. Initially, cardiomyocytes hypertrophical changes appear, and also changes of the edematous matrix. Then, the nuclear lesions, the apoptosis processes and the hypoxic alterations increased to cytolysis. In succession, after the matrix edema was organized, immune lesions and myocardial sclerosis processes appeared. Out of the various lesions, we mention: the sarcoplasmatic fuchsinophilia and alcianophilia, the glycogenic depletion, the decrease of SDH, LDH reactions, the increase of the cytolitic enzymes, especially of the kathepsine B. Out of the ultrastructural changes, we mention: the mitochondrial, sarcotubular lesions and, the lysosomial activations, the interstitial organelles, including the natriuretic granules, synthesized in the ventricular myocytes, details regarding the matrix changes with tendency to fibrosclerosis.

Morphological and immunohistochemical features of tumor infiltrating lymphocytes (TIL) in carcinomas. Note I.

Some malignant tumours like testicular seminomas and ovarian dysgerminomas or medullar carcinoma of the breast, present an unusually lympho-histiocytic (TIL) rich stroma. Many reports have concluded that the prognosis for these patients is correlated with the intensity of TIL. Recently, some analyses consider that tumour-host interactions have a significant prognostic role in many other neoplasms. The presence of TIL may be a sign of less aggressive behaviour of some epithelial neoplasm like gastro-intestinal, pulmonary, mammary, urinary or cutaneous carcinomas (Wilson et al.). The aim of this study is a morphological and immunohistochemical (IHC) characterisation of the lympho-histiocytic populations of TIL in some gastro-intestinal and mammary carcinomas. The two localisation were chosen for their different contact with the exogene antigens and their possible different type of host's immune response.

Electronmicroscopic observations regarding the presence of natriuretic granules in the ventricle of patients with cardiopathies.

As part of studies regarding the human myocardium biology in hereditary and acquired cardiopathies, we showed the presence of natriuretic granules (NG) not only in atriums but also in ventricles, in ten of our cases with intraoperative myocardium biopsies. Ultrastructurally, the natriuretic granules occur in lesions produced by hemodynamic and consecutively hypoxic disturbances, being present at the ventricular level, too, both near the Golgi apparatus that secrets them, and diffusely, in cardiomyocytes, beneath the altered organelles and phagolysosomes. Their aspect in similar in cardiac malformations (DSIA. Fallot tetralogy) and in mitral valvulopathies, their abundance being in connection with congestive heart failure (CHF). Although predominant in CHF class I and II, they occur also in the severe decompensations class III and IV, being perhaps felt at the cardiac level as they are released in the blood. This phenomenon is expressed by electronmicroscopic presence of natriuretic granules in the subsarcolema and the increased plasma level of natriuretic peptide, according to biochemical findings reported in the literature. They produce a vasodilating, diuretic, natriuretic effect, contributing to blood pressure regulation and testifying in the neuroendocrine role of human myocardium.

Morphophysiopathologic studies on the biology of myocardium in acquired valvulopathies.

Myocardial lesions due to various valvulopathies were sampled during open heart surgery under extracorporeal circulation, and histologically, histoenzymologically and ultrastructurally investigated. Various cardiomyocitary and interstitial nonspecific progressive lesions, due especially to hemodynamic and hypoxic alterations, were identified. During the pathogenic stages, we remarked the onset and chronic evolution of perivascular and interstitial oedema, enhanced by a reduced lymph draining. The progressive processes of hyalinization, sclerosis and interstitial fibrosis were subsequent to the interstitial matrix modification. These processes accentuate the myocardial hypoxic lesion due to the nutritional and gaseous exchange alterations. The infrastructure, mitochondrial enzyme equipment, sarcoplasma and tubular network lesions, as well as the intramyocitary oedema that destroys the sarcomeric structure, ended with the activation of lysosome and lysosomal enzymes, giving rise to consequent cytolytic foci. Therefore, the process was extending. In this context, we remarked at the electronmicroscopic examination ventricular natriuremic granules, that are rarely mentioned in literature.

Histoenzymological and ultrastructural studies of the macrophage-type pulmonary cell modifications under different chronic stasis conditions.

The optical histoenzymological and electronomicroscopical investigations were carried out en 90 pulmonary biopsies taken from patients under different chronic stasis conditions. To investigate macrophage-type cells we studied the reactions of acid phosphatase and non-specific esterase like enzyme markers, indicating the metabolic state activation degree of this cellular compartment. The ultrastructural examinations revealed the existence of two types of activated pulmonary macrophages. The former is the alveolar macrophage with a prevailing phagolysosome apparatus that pleads for phagocytic function and the latter is the interstitial macrophage with subcellular aspect corresponding to the synthesis and secretion function. The presence of these activated alveolar and interstitial macrophages, as well as their extremely increased number, plead for their direct participation in the appearance and aggravation of the morphophysiopathological disorders in the chronic stasis lungs.

Histoenzymological and ultrastructural investigations regarding the role of the endogenous peroxidase at the pulmonary level in cardiogenic pulmonary hypertension.

We found optically and ultrastructurally endogenous peroxidase in 90 intraoperatory pulmonary biopsies taken from patients with valvular cardiopathies and different degrees of pulmonary hypertension. The enzymatic activity increases proportionally to the pulmonary hypertension and it reaches its maximum in severe cases of hemosiderosis. Ultrastructurally we found two reaction types namely: 1) in peroxisomes of circulating monocytes turning into tissue ones, in medium or slight pulmonary hypertension (PHT) and 2) (as granular reactions on the nuclear membrane and endoplasmic reticle in alveolar macrophages, granular pneumocytes in aggravating and severe medium PHT. The presence of both these two types of reaction in the same cell demonstrates the passing stages from circulating macrophages to tissue ones attesting a transitional process of the phagocytic cells.

Histoenzymological and ultrastructural research of myocardial mitochondria in cardiac insufficiencies.

We performed histoenzymological and electron-microscopic investigations of 40 myocardial biopsies taken during open heart surgery under extracorporeal circulation (ECC) from patients with valvular cardiopathies and septal interatrial defects with various degrees of congestive cardiac insufficiency (CCI). The changes occurring in the topochemistry and intensity of the enzymatic reactions used as markers for mitochondria, namely SDH and LDH, demonstrated the existence of a correlation between the decrease of reactions of the CCI degree. The ultrastructural aspects of the mitochondrial changes attested the precocity and severity of damage in these organelles even from initial moments of hypoxia.

Morphophysiopathological correlations in pulmonary hypertension of cardiac origin.

100 patients with congenital malformation and acquired cardiac diseases were studied on preoperatory pulmonary biopsies to estimate the pulmonary hypertension by correlating the data obtained by modern multidisciplinary investigation: histological, histoenzymological, ultrastructural, physiological and clinical techniques. The morphological pictures show a range of vascular lesions similar to those found in Heath and Edwards' classification and they are especially thickenings of the intima, hypertrophies of the media, narrowings of vascular lumen and later some plexiform lesions and hemosiderosis. Progressive fibrosis processes as well as immune pulmonary reactions were made evident. The electron microscopical examinations provided new data regarding the thickening of the capillary and alveolary basal lamina, the activity of pneumocytes and macrophages that were also certified from the histoenzymological point of view by increase of peroxidases and acid phosphatases activity. The investigation carried out proved the importance of the cardiac preoperatory catheterism in order to establish the grade of the pulmonary hypertension while the concordance index is more increased than in the cases that were investigated only by non-bleeding techniques.

Histochemical and ultrastructural alterations of lysosomes and acid phosphatase reaction in human chronic hypoxic myocardium.

Histochemical and ultrastructural alterations of lysosomes and acid phosphatase reaction were assayed in myocardial human specimens from patients with some acquired and congenital cardiac affections and cardiac failure class II-IV (after NYHA classification). The optic and electron microscopic study of the acid phosphatase (the main lysosomal marker) activity reveals that the intensity and the distribution of the reaction were correlated with the degree of heart failure, being maximum at the IVth degree. The electron microscopic analysis of the lysosomal vacuolar apparatus emphasizes various changes also correlated with the intensity of hypoxia.

Histochemical reactions of myocardial proteases during open heart surgery.

Histochemical analysis of some lysosomal and sarcoplasmic proteolytic enzymes was assayed in human myocardial biopsies taken from 26 cardiopathic patients subjected to open heart operations, under extracorporeal circulation and protection with cardioplegic solution and hypothermia. The investigated myocardial proteases were: cathepsin B, cysteine aminopeptidase, acid gelatinases, trypsin-like endopeptidase, chymotrypsin-like endopeptidase and neutral gelatinases. The effects of surgical interventions appreciated by comparing the myocardium fragments harvested before, and at various intervals after aorta clamping (6-90 minutes) revealed disorders in the activity and compartmentalization of all the investigated proteases, whose histochemical reactions increased between 10 and 20 minutes after aorta clamping and manifested a lowering tendency with sarcoplasmic diffusion and extracellular release at longer periods than 20 minutes. The early activation of the neutral proteases and their sarcolemmal expression even before 10 minutes after aorta clamping, suggested the involvement of the nonlysosomal proteases in the first proteolytic events implied in the molecular membrane damage of the myocardial fibre. Sequential proteolytic cascades of abnormal neutral and acid proteases were emphasized as possible mediators and effectors of molecular and subcellular damages suffered by the myocardial fibers during the open heart operations, even under cardioplegic and hypothermic protection.

Histochemical investigation of myocardial proteases in heart anoxia, under protection with cardioplegic solution and protease inhibitors.

We studied the histochemical behaviour of neutral non-lysosomal and acid lysosomal proteases of the myocardium in heart anoxia by aorta-clamping, using an experimental model alike to the open heart surgery conditions. After one hour of blood deprivation we found destabilization, conglomeration, diffusion and extracellular release of the histochemical reactions for neutral BANA-reactive proteases, cathepsin B, acid and neutral gelatinases in monkey, rat and mice myocardium, in connection with lytic ultrastructural lesions. Intracardiac perfusion with cardioplegic solution exerted a partial protective effect which is improved by adding Trasylol, a protease inhibitor. Administration of Trasylol by intravenous injections preceding aorta clamping, or/and by intracardiac perfusion after aorta clamping, normalized the histochemical reactions of proteases together with the ultrastructural organization of the myocardial fibres. Acting especially by the inhibition of membrane serine proteinases, Trasylol may intercept membrane permeabilization events, impairing the proteolytic cascades triggered by anoxia.