[Carotid ultrasound: prevention of heart disease and mortality on haemodialysis]. / La ecografía carotídea es útil para predecir enfermedad coronaria y mortalidad en pacientes en hemodiálisis.

INTRODUCTION: Cardiovascular disease and other complications of atherosclerosis are the most common cause of death in patients with chronic renal failure in maintenance hemodialysis (MHD). Carotid ultrasonography is a simple non-invasive tool to investigate the vascular system, by means of intima media thickness (IMT) measurement and carotid wall calcifications. OBJECTIVE: To determine IMT and the presence of plaques, and their possible clinical relationships; finally we tried to investigate whether they would predict cardiovascular morbidity and mortality in patients in MHD. METHODS: We studied 60 MHD patients (age 68 +/- 13 years, 48% male, 50% diabetics, time on MHD 32 +/- 11 months) and a control group of 274 people matched for age and sex. Follow-up period was 66 +/- 13 months. MEASUREMENTS: Demographic and clinical data, serum levels of homocysteine (tHcy), folic acid (FA) and B6 and B12 vitamins. IMT was measured by high-resolution B-mode ultrasonography. RESULTS: IMT was higher in MHD patients than in those in the control group (0.947 +/- 0.308 vs 0.619 +/- 0.176 mm; P < 0.001). IMT was related with age (r = 0.268; P = 0.038), diabetic (r = 0.650; P < 0.001) and hypertensive condition (r = 0.333; P = 0.012), but not wih lipids, tHcy or FA. Patients who suffered from coronary artery disease, peripheral artery disease or stroke had higher IMT than those without those events (1.156 +/- 0.371 vs 0.875 +/- 0.285 mm; P < 0.001; 1.205 +/- 0.374 vs 0.911 +/- 0.231 mm; P = 0.007; 1.195 +/- 0.264 vs 0.844 +/- 0.251; P < 0.001 respectively). Something similar occurred with the presence of plaques. During the follow-up period 36 patients died (60%), 67% of them due to cardiovascular causes. IMT was higher in patients who died than those who survived (1.020 +/- 0.264 vs 0.858 +/- 0.334 mm; P = 0.044). The survival rate during the observation period was significantly lower in the final IMT fourth (20%) than in the first (72%) (P = 0.014). The presence of carotid plaques was an independent predictor of cardiovascular mortality. CONCLUSIONS: These findings suggests that measurement of carotid IMT and the presence of wall plaques are useful tools to predict cardiovascular events and mortality in patients in MHD.

Biological and prognostic role of acid cysteine proteinase inhibitor (ACPI, cystatin A) in non-small-cell lung cancer.

BACKGROUND: Acid cysteine protease inhibitor (ACPI) is an intracellular protein, often linked to neoplastic changes in epithelium and thought to have an inhibitory role in malignant transformation. AIM: To analyse the expression and prognostic role of ACPI in non-small-cell lung cancer (NSCLC). METHOD: Histological samples from 199 patients with resected NSCLC were stained immunohistochemically for the expression of ACPI in normal and preneoplastic bronchial epithelium, and in various types of lung carcinomas. RESULTS: A normal bronchial epithelium showed positive staining for ACPI in the basal cells, whereas the upper two-thirds of the dysplastic epithelium was ACPI positive. High staining for ACPI was found in 74% (91/123) of squamous-cell carcinomas, whereas 16% (8/49) of adenocarcinomas and 30% of (8/27) large-cell carcinomas showed the high expression of ACPI (p<0.001). Among squamous-cell carcinomas, low expression of ACPI was correlated with poor tumour differentiation (p=0.032). In the whole tissue, reduced expression of ACPI was associated with tumour recurrence (p=0.024). In overall survival (OS) and disease-free survival (DFS) analyses, the histological type of the tumour (both p<0.001) and stage of the tumour (p=0.001, p=0.013, respectively) were related to patient outcome. Low expression of ACPI in tumour cells was associated with poor OS and DFS (p<0.041, p=0.004, respectively). In multivariate analysis, ACPI did not retain its prognostic value, whereas the traditional factors were the most important prognostic factors. CONCLUSIONS: ACPI expression is linked with the malignant transformation of the bronchial epithelium and predicts a risk of tumour recurrence as well as poor rate of survival for the patients. However, ACPI does not have any independent prognostic value in NSCLC.

Human spleen cysteineproteinase inhibitor. Purification, fractionation into isoelectric variants and some properties of the variants.

The papain inhibitor from human spleen was purified by extraction in isotonic sucrose, acetone fractionation, papain-Sepharose affinity chromatography and gel filtration on Sephadex G-50. The purified inhibitor was fractionated by electrofocusing into four major isoelectric variants with pI values of 4.7, 5.0, 6.0 and 6.5. These variants can be classified into two groups: the acidic type, comprising the variants with pI 4.7 and 5.0, and the neutral type, comprising the variants with pI 6.0 and 6.5. The following properties distinguish the two types: 1. Immunological properties: antibodies raised against either of the neutral variants precipitated both of these, but not the acidic variants. The antiserum against the human epidermal cysteineproteinase inhibitor precipitated the acidic variants, but not the neutral variants. 2. Molecular size: two-dimensional electrophoresis of the purified inhibitor gave molecular weights of 11400 for the acidic variants and 12000 for the neutral variants. The pI 6.0 variant contained two compounds with molecular weights of 12000 and 12800. 3. Enzyme spectrum: human cathepsin B was inhibited by the acidic type, while the neutral type was a poor inhibitor. Both types inhibited cathepsin H, papain, ficin and bromelain, although the inhibition of bromelain did not exceed 70%. Human cathepsin D, bovine trypsin and chymotrypsin and porcine elastase were not inhibited by either type.

The low-molecular-weight SH-protease inhibitor in rat skin is epidermal.

The epidermis and dermis of rat skin were separated and the presence of the high-molecular-weight SH-protease inhibitor I1 and the low-molecular-weight inhibitor I2 in both was studied. Gel filtrations of the extracts revealed that 97% of the epidermal inhibitor activity was due to I2 and 89% of the dermal activity to I1. The presence of I2 mainly in the epidermis extract was confirmed by immunodiffusion of specific rabbit anti-I2 serum against purified I2, epidermis and dermis extracts, and rat serum. Most of the immunoreactive protein was seen in the epidermis extract, traces in the dermis extract and none in the rat serum. I2 was localized in rat skin by indirect immunofluorescence using rabbit anti-I2 serum and fluorescein isothiocyanate conjugate of goat anti-rabbit immunoglobulins. Intense fluorescence, much brighter than in the controls treated with rabbit non-immune serum, was seen in the epidermis, being most pronounced in the cytoplasms of cells in the granular layer. The weak fluorescence of the hair follicles, sebaceous glands, connective tissue cells and fibres was unspecific and was also seen in the controls. In view of its epidermal location, the name epidermal SH-protease inhibitor is suggested for I2.

Partial purification and some properties of a new papain inhibitor from psoriatic scales.

A new papain inhibitor was purified from psoriatic epidermal scales using gel chromatography and anion exchange chromatography. The purified protein inhibited papain and ficin but not cathepsin B, cathepsin H, trypsin, or chymotrypsin. Isoelectric focusing revealed 3 major inhibitor variants with pI's of 7.3, 6.9, and 6.5. A Mr of 38,000 was obtained by a gel chromatographic method for the crude inhibitor. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr values of the isoelectric variants were: 43,000 for the variant pI 7.3, 43,000 and 35,000 for the variant pI 6.9, and 34,000-35,000 for the variant pI 6.5. An antiserum of the inhibitor was used to locate the inhibitor in the psoriatic and normal epidermis. In psoriatic epidermis, the inhibitor was found in the peripheral cytoplasm of spinous cells and in the scale. In normal epidermis, the staining was seen only in orifices of hair follicles. An inhibitor with similar size and antigenic properties to that isolated from the psoriatic scales was demonstrated in extracts made from the whole-thickness epidermis but not in extracts from the healthy epidermal scales, the dermis, the liver, the spleen, or the blood serum.

Primary structure of bovine cathepsin S. Comparison to cathepsins L, H, B and papain.

The primary structure of bovine cathepsin S was determined by combining results of protein and peptide sequencing with the sequence deduced from nucleic acid sequencing. Using polymerase chain reaction (PCR) technology, cDNA clones commencing at amino acid 22 of the mature enzyme and continuing through the 3' untranslated region of bovine cathepsin S mRNA were isolated and sequenced. The open reading frame in these overlapping clones correctly predicts the determined amino acid sequence of 13 tryptic peptides derived from purified bovine spleen cathepsin S. The deduced amino acid sequence shows that mature bovine cathepsin S consists of 217 amino acids corresponding to a molecular weight of 23.7 kDa. Cathepsin S belongs to the papain superfamily of lysosomal cysteine proteinases and shares 41% identity with papain. Amino acid sequence identities of bovine cathepsin S to human cathepsins L, H, and B are 56%, 47% and 31% respectively.

Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoroimmunoassay.

A time-resolved fluoroimmunoassay was developed for the detection of 3 human low molecular weight cysteine proteinase inhibitors, ACPI (cystatin A), NCPI (cystatin B), and gamma-trace (cystatin C). Polystyrene tubes or polystyrene microtitration strips were used as solid phase. The rabbit anti-inhibitor immunoglobulins were used as the capture antibody, and, when labelled with europium, also as the detector antibody. The threshold sensitivity of the tests was 0.1 ng/ml for NCPI and 1 ng/ml for the others. All the 3 cysteine proteinase inhibitors, ACPI, NCPI, and gamma-trace, were detected in pooled serum samples of patients with kidney failure. gamma-Trace seemed to be quantitatively the major and ACPI the minor inhibitor. No other low molecular mass cysteine proteinase inhibitor was detected after isoelectric focusing of the 12 kDa area of gel filtered human serum.

Concentrations of lysosomal cysteine proteases are decreased in renal cell carcinoma compared with normal kidney.

Renal cell carcinoma contains significantly lower concentrations of the lysosomal cysteine proteases, cathepsins B, C, H, L and S, than does normal kidney, as shown by several methods, such as activity determination, enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry. The same low levels of enzyme activity and concentration have been determined in renal cell carcinoma metastases in the lung. Our results on the decreased concentration of cysteine peptidases at the protein level would seem to conflict with earlier results on an increased concentration of the cathepsin L mRNA in renal cell carcinoma.

Antigenic expression of cathepsin B in aged human brain.

The lysosomal thiol proteinase, cathepsin B, has been localized in different regions of aged human brain by use of the peroxidase-antiperoxidase technique. Cathepsin B-immunoreactive material was detected in multiple neurons of human hippocampus, neocortical area A 10, prefrontal gyrus and nuc. basalis of Meynert as well as in single white matter astrocytes. In brains of Alzheimer disease-affected subjects cathepsin B was revealed in neuritic plaques too. Possible functional consequences with regard to normal aging, neuropeptide metabolism and pathological changes are discussed.

Cystatin A-like immunoreactivity is widely distributed in human brain and accumulates in neuritic plaques of Alzheimer disease subjects.

The cellular localization of cystatin A, an endogenously occurring inhibitor of lysosomal thiol proteases (cathepsins B, H, L and S), was studied immunohistochemically in human postmortem brain using the peroxidase-antiperoxidase method. Both polyclonal and monoclonal antibodies to cystatin A were employed. Western blot analysis revealed one molecular form of the inhibitor in human brain extracts. Its molecular weight was about 13,000. Immunostaining appeared in a sizeable population of neurons and a few cells surrounding cerebral blood vessels (pericytes). In Alzheimer disease subjects cystatin A was found in many neuritic plaques. Possible functional consequences with regard to a role of cystatin A in the inhibition of the Alzheimer amyloid precursor protein (APP)-clipping enzyme, cathepsin B, are discussed.

Production of acid and neutral cysteine-proteinase inhibitors by a cultured human skin epithelium cell line.

Human skin epithelial-like cells (NCTC-strain 2544) were grown in RPMI-1640 medium supplemented with foetal calf serum for up to 2 weeks. The culture medium and extracts made from the cells were subjected to gel-filtration chromatography in a Sephacryl S-200 column for fractionation of the proteins. The fractions were assayed for acid and neutral cysteine-proteinase inhibitor (ACPI, NCPI) using time-resolved fluoroimmunoassay and radioimmunoassay, and the cysteine-proteinase-inhibiting activities were assayed using papain. Free NCPI, i.e. a molecule with isoelectric variants at pHs 6.0 and 6.5, which has an Mr of around 12,000 and is capable of inhibiting papain, was detected both in the culture medium and in the cells. Immunodiffusion studies revealed its immunological identity with human spleen-derived NCPI. The amount of NCPI increased during the incubation period. ACPI--characterized as a molecule having an isoelectric point of 4.9, an Mr of about 12,000, papain-inhibiting capacity and antigenic reactivity with spleen-derived ACPI--was not detected in the culture medium. It was, however, detected in the cells after 2 weeks in culture. These data prove that ACPI and NCPI are synthesized by the NCTC-2544 cells under the present culture conditions.

A 43-kDa papain inhibitor produced by a cultured human epidermal cell line.

Epidermis-derived cells (NCTC 2544) were cultured and the proteins of the culture medium, as well as of the cells, were fractionated by gel-chromatography. The fractions were analyzed for their papain-inhibitory capacity and for the presence of so-called 43-kDa papain inhibitor. A papain inhibitor was identified with molecular weight and immunological characteristics similar to the original 43-kDa inhibitor that was isolated from psoriatic scales. The result proves that NCTC-2544 cells can produce the so-called psoriasis inhibitor under culture conditions.

Cysteine proteinase inhibitors in psoriatic epidermis.

Human psoriatic epidermis and scales were demonstrated to contain two antigenically separate cysteine proteinase inhibitors, one acidic with an isoelectric point of 4.7-5.0 (ACPI) and one neutral with an isoelectric point of 6.0-6.5 (NCPI), while normal epidermis contains only ACPI. The total papain (cysteine proteinase) inhibiting activity of the psoriatic epidermis as calculated per mg protein was higher than that in normal epidermis. Both ACPI and NCPI were localized immunocytochemically, mainly in the highest spinous cell layers with less activity in the parakeratotic cells and lower layers of spinous cells. Basal cells were essentially negative.

Localization of cathepsin H and its inhibitor in the skin and other stratified epithelia.

The rat-skin-derived cysteine proteinase, so-called BANA-hydrolase, which is capable of hydrolysing benzoylarginine naphthylamide and leucine naphthylamide was shown to be immunologically identical to cathepsin H purified from rat liver. The enzyme was immunocytochemically localized in the basal cell layer of rat epidermis. A natural inhibitor of cathepsin H with a molecular weight of about 13,000 was mainly localized in the keratinizing cell layers and showed only a weak reaction in the basal cells. Thus, cathepsin H appears to be a characteristic feature of the proliferating cell layer, whereas the cysteine-proteinase inhibitor is a characteristic feature of keratinizing cells.

Sound spectrographic analysis of pain cry in preterm infants.

Sound spectrographic cry analysis was performed on 302 cries of 48 preterm infants born at 30-37 gestational weeks. The cries were recorded during the first week of life and thereafter weekly until the infants were discharged. The control series comprised 54 cries from 27 fullterm healthy infants. The results showed that the cries of the smallest prematures compared with the controls were shorter, more high-pitched, and included bi-phonation and glide more often. The cry characteristics changed with increasing conceptual age and the older the child the more the cry pattern resembled that of the fullterm. The cries of the preterm infants when they had reached 38 conceptual weeks were similar to those of newly born fullterm infants. The results indicate that the gestational age should be taken into consideration in cry analysis.

Epidermal SH-protease inhibitor in human neoplasms and their metastases.

The presence of the human epidermal SH-protease inhibitor in human tumours of different types was examined using double radial immunodiffusion against specific antisera to the inhibitor. The immunoreactive protein was found to be present in all the tumours with criteria of epidermoid carcinoma identifiable microscopically. Most small cell anaplastic, fusiform, and polygonal bronchial carcinomas were also found to contain immunoreactive protein. If the primary tumour contained the inhibitor, all metastases examined were also found to contain it. The identity of the inhibitors in epidermoid carcinomas and in epidermis was confirmed by immunoelectrophoretic and gel chromatographic methods. The results suggest that the epidermal SH-protease inhibitor is a property closely associated with squamo-epithelial tissue differentation, which also appears in neoplastic squamous epithelium.

Effects of vanadium on the upper respiratory tract of workers in a vanadium factory. A macroscopic and microscopic study.

An epidemiologic cross-sectional case-history study on the injurious effects of vanadium was carried out among the workers of a vanadium factory. The upper respiratory tract of 63 male workers exposed to vanadium dust was examined macroscopically and microscopically, and the findings were compared with those of a reference group of workers who were exposed to inert dust only. The groups compared were of similar ages and had similar smoking habits. Nasal smears and sputum cells were studied microscopically, and biopsies for histological study were taken from the nasal mucosa. The biopsies from the vanadium workers showed a significant increase in the number of plasma and round cells, and the histological picture was almost characteristic. There were no increased numbers of secretion eosinophils or other signs indicative of allergic inflammation.

Tumours and sudden death.

A series of 27 sudden deaths caused by tumours, amounting to 1.2% of all deaths in the 5-year period studied, is found to include 9 cases where the death had also been unexpected, since the fatal tumour had gone undiagnosed. Four of these cases had had no preceding symptoms. Males were predominant (19 males vs. 8 females), and all the sudden unexpected deaths were males. The age range was 24 - 86 years for the males and 45 - 83 years for the females. The most common tumour was bronchial epidermoid carcinoma, which caused sudden death by massive haemorrhage into the lungs or externally. In the latter case the scene of death at home resembled a violent death. Other tumours found were mostly gastric or oesophageal epidermoid carcinomas and adenocarcinomas of the kidney, pancreas, ovary and colon.

A protein reminiscent of the epidermal SH-protease inhibitor occurs in squamous epithelia of man and rat.

The occurence of the human and rat epidermal SH-protease inhibitors in various human and rat tissues was studied by double radial immunodiffusion against specific antisera to the inhibitors. An immunoreactive protein was found in the extracts prepared from human and rat epidermis and from eosophageal and vaginal squamous epithelia, and from rat pro-ventricular squamous epithelium. No immunoreactive protein was found in man or rat in any other of their tissues, studied by us. The results strongly suggest that a protein reminiscent of the human or rat epidermal SH-protease inhibitor is present in squamous epithelia but not in other tissues. The identity of the epidermal inhibitor and the immunoreactive protein in the other squamous epithelia was confirmed by immunodiffusion, immunoelectrophoresis and gel chromatography, and by immunoinhibition of the papain inhibiting activity of the human epidermal and oesophageal inhibitors by gammaglobulins separated from antiserum to the human epidermal inhibitor.

Localization of the human SH-protease inhibitor in the epidermis. Immunofluorescent studies.

Human epidermis contains a low molecular weight SH-protease inhibitor (Human Epidermal Inhibitor = HEI), whose epidermal localization was performed with the indirect immunofluorescence method. The fluorescence was most intensive in the cytoplasms of epidermal cells, often occurring perinuclearly. The fluorescent material in the frozen sections was often finely granular and occasionally extended outside the cytoplasm, while the fluorescence in fixed sections was more uniform, but weaker. Stratum basale generally stained poorly or not at all, as did also stratum lucidum. Stratum corneum stained fairly intensively throughout. In addition to fixation, the outcome of staining was also affected by the thickness of the epidermis, particularly stratum corneum. The significance of this inhibitor for the differentiation of epidermal cells and the keratinization of epidermis has therefore been discussed, and the authors assume it to be of considerable significance in these processes.