The kinin system in patients with systemic lupus erythematosus exhibiting mucocutaneous lesions: a clinical study.

In the present study, we evaluated the kinin system components in the plasma of patients with systemic lupus erythematosus exhibiting mucocutaneous lesions. Fifteen women with active cutaneous lupus (P) and 15 normal healthy women (C) were studied. Low molecular (LKg) and high molecular (HKg) weight kininogen were determined by ELISA (expressed microg Bk/ml). The activities of tissue kallikrein (TKal), plasma kallikrein (PKal) and kininase II were assayed by their action on selective substrates. Statistical analysis was performed using the Mann-Whitney test. The patients presented increased plasma levels of LKg (P = 2.98, C = 0.79) and HKg (P = 1.78, C = 0.5) associated with the increased activity of PKal (P = 2.50, C = 1.63 U/ml), TKal (P = 1.87, C = 1.30 microM pNa/ml) and kininase II (P = 1.50, C = 0.51 microM Hys-Leu/ml), when compared to the values observed in the control group (P < 0.0001 for each comparison). Thus, the increased concentration of all parameters of the kinin system in these patients indicate an overactivity of the kinin system in the acute phase of lupus, corroborating with the participation of these mediators in lupus pathogenesis.

Chronic antigen ingestion protects ovalbumin sensitized mice from severe manifestation of Leishmania major infection.

In the present work, the development of experimental leishmaniasis was examined in sensitized BALB/c mice that were chronically fed with antigen. After an oral challenge with egg white solution, the ovalbumin (Ova)-sensitized mice showed an increase in serum anti-Ova IgE and IgG1 antibodies. Lesions induced by Leishmania major infection were reduced by the ingestion of Ova in sensitized mice, as assessed by reduced footpad growth, lower parasite loads and improved pathological outcome compared to sham sensitized mice. Moreover, such findings were connected to a shift to a Th1 response involving higher IFN-gamma production and serum levels of IgG2a anti-Leishmania antigens. The data appear to corroborate the suggestion that chronic ingestion of an antigen by sensitized mice modulates the immunological system through a shift in cytokine release, exhibiting a healing response and resistance to L. major infection.

Evaluation of leucocytes from sputum samples of pulmonary tuberculosis patients using flow cytometry.

SETTING: Information about the sputum cells of pulmonary tuberculosis (PTB) patients is scarce. The analysis of sputum cells using optical microscopy (OM) is a well-established method, but it has some serious limitations. OBJECTIVE: To establish a new flow cytometry (FC) protocol for the leucocyte evaluation of sputum samples from PTB patients. DESIGN: A new FC protocol using 0.1% dithiothreitol and 0.5% paraformaldehyde was developed to fluidise sputum samples and kill Mycobacterium tuberculosis, respectively, to allow the analysis of sputum samples collected from TB patients. The protocol was validated by comparing it with OM, and the cellularity of 30 sputum samples from patients with PTB was evaluated. RESULTS: The comparison between leucocyte subsets analysed using OM and FC showed agreement. Immunophenotyping of leucocytes from sputum samples showed that neutrophils (95.7%) comprised the largest proportion of sputum cells, followed by monocytes/macrophages (2.6%) and lymphocytes (1.6%). Among the total T-lymphocytes (100%), 12.3% were T-helper cells, 24.1% were cytotoxic T-cells and 62.9% were gamma/delta T; none of the T lymphocytes had the CD4+/CD8+ phenotype. CONCLUSION: FC is a useful method for evaluating the different subtypes of leucocytes present in the sputum samples of PTB patients.

Rat high-molecular-weight kininogen: purification, production of antibodies and demonstration of lack of immunoreactive kininogen in a strain of brown Norway rats.

High-molecular-weight kininogen was purified to apparent homogeneity from Wistar rat plasma by a two-steps chromatographic procedure. 3 mg of kininogen were obtained from 205 ml of plasma. The purified high-Mr kininogen had a bradykinin content of 10.2 micrograms bradykinin equivalents/mg protein. Under denatured and reduced conditions it gave a single band on polyacrylamide gel electrophoresis corresponding to an apparent molecular mass of 110 kDa. Antibodies obtained against rat high-Mr kininogen gave a single precipitation line when tested against rat plasma in double immunodiffusion and in crossed immunoelectrophoresis. Although rat high-Mr kininogen possesses physicochemical properties (molecular mass, kinin content per molecule and amino acid composition) similar to human high-Mr kininogen, its antibodies do not cross-react with human, monkey or rabbit plasma, indicating major interspecies differences in the structure of the molecule. Immunoreactive kininogen of Wistar rats was identical to that of Brown Norway rats from a strain bred in Orleans, France (BN/Orl). However, plasma from a strain of Brown Norway rats bred in Leuven, Belgium (BN/Kat), reported to be deficient in a kinin precursor (Damas, J. and Adam, A. (1980) Experientia 36, 586-587), did not contain immunoreactive material discernible by double immunodiffusion or crossed immunoelectrophoresis.

Increased levels of new spasmogenic substances released by trypsin from plasma of hypertensive rats.

The present study was undertaken to evaluate the participation of the kallikrein-kinin system in the normalization of blood pressure after release of the clip in one-kidney, one clip hypertensive rats ( 1K1C ). Kininogen was determined before and after unclipping by tryptic digestion of denatured rat plasma, and spasmogenic activity was measured with isolated guinea pig ileum. In contrast to human plasma for which bradykinin (BK) is the only trypsin-releasable spasmogenic substance ( TRSS ), rat plasma contains non-BK TRSS (Fractions P1 and P2) as well as BK. Fractions P1 and P2 were separated from BK by SP-Sephadex chromatography. An increase of total TRSS was demonstrated 60 days after clipping and reached a maximum at approximately Day 75, which was two times that of the normotensive controls (NC). The level of total TRSS did not change after unclipping . The increased level of TRSS in the hypertensive state confirmed the observations of other investigators who reported increased kininogen levels but who could not distinguish between BK and non-BK TRSS because bioassays were performed without prior chromatographic separation of the spasmogenic activities. Fractions P1 and P2 were present in the TRSS of both 1K1C and NC plasma, but were two to six times higher in 1K1C and thus probably accounted quantitatively for the increased TRSS in 1K1C . The data suggest that in the hypertensive state there is an alteration in the relative amounts of some plasma proteins that contain non-BK TRSS within their amino acid sequences. Fractions P1 and P2 also contain potentiating peptides and have not yet been purified to homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

Kininogen and prekallikrein increases in the blood of streptozotocin-diabetic rats are normalized by insulin in vivo and in vitro.

Twelve days following treatment with 50 mg/kg streptozotocin (STZ), male rats were diabetic, with a three-fold increase in blood glucose (P<0.001) and increased plasma bradykinin (BK) kininogen reserves of [high-(HK)- and low- (LK)-molecular-weight kininogens,+162%, P<0.01 and +63%, P=0.05, respectively], as determined by bioassay of BK released by trypsin from these precursors under standardized conditions. Administration of a single dose (10 U/kg i.v.) of regular insulin decreased plasma HK and LK to near non-diabetic values. Within 24 h these values had returned to levels characteristic of uncorrected diabetes. Prekallikrein (PK), the precursor of plasma kallikrein, an enzyme which releases BK from HK, was increased by 63.4% (P<0.05) in STZ-diabetes, but dropped to near normal levels following insulin treatment. Incubation of whole blood of normal or diabetic rats with 0.02-0.2 mU/ml regular insulin for 10 min at 37 C, decreased HK (P<0.01) and PK (P<0.05) and led to the appearance (P<0.05) of Arg-Pro-Pro-Gly-Phe, a partially stable product of BK metabolism, detected in the incubation media by an enzyme-linked immunosorbent assay (ELISA). Incubation of cell-free plasma insulin had no effect on these parameters, suggesting that blood cells, possibly neutrophils, are required by insulin for the activation of plasma PK to kallikrein leading to BK release. Insulin may be a factor modulating BK formation; its reduction in diabetes may explain increases of plasma kininogen and PK observed in this condition.

Kinin system in lupus nephritis.

There are few studies regarding the evaluation of the kinin system in patients with systemic lupus erythematosus (SLE). In this study, we evaluated the plasma levels of high-molecular weight kininogen (HKg), low-molecular weight kininogen (LKg) and plasma kallikrein; the plasma activity of tissue kallikrein and kininase II, and urinary kallikrein and kininase II activities in patients presenting with active lupus nephritis. A total of 30 patients (29 women) aged 21-62 years (median = 39) and 30 controls matched to the patients for sex and age were studied. Patients presenting with other underlying diseases or using drugs, which could interfere with the kinin system, were excluded. HKg and LKg levels were indirectly evaluated by ELISA. Plasma kallikrein, tissue kallikrein, and kininase II were evaluated by their enzymatic activity on selective substrates. The Mann-Whitney test was used for statistical analysis. HKg, LKg and plasma kallikrein levels were significantly increased in patients (p < 0.001, for each comparison). Similarly, tissue kallikrein and kininase II activities were significantly increased in plasma and urine of patients (p <0.001, for each comparison). In urine, the activities of tissue kallikrein and kininase II were at least seven times higher than those seen in the plasma of patients. These results indicate that the kinin system is involved in the acute manifestations of lupus nephritis. Kinins may facilitate immunecomplex deposition and may induce the release of other pro-inflammatory mediators, including cytokines actively involved in the pathogenesis of lupus nephritis.

Increased plasma levels of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha in patients moderately or severely envenomed by Tityus serrulatus scorpion sting.

Scorpion envenomation is a common medical problem in many countries and an important cause of morbidity and mortality, especially among children. The plasma levels of pro-inflammatory (IL-1beta, IL-6, IL-8 and TNF-alpha) and anti-inflammatory (IL-10) cytokines were measured in individuals stung by Tityus serrulatus (Ts) scorpions. According to clinical manifestations patients were classified, as defined by the Brazilian Ministry of Health, as having mild (n=15, mean age=42.2 years), moderate (n=8, mean age=26 years) or severe (n=4, mean age=14 years) envenomation. Blood samples were taken immediately (T1) and 6h (T2) after admission to the hospital. Eighteen age-matched healthy volunteers were used as control. TNF-alpha, IL-1beta, IL-6 and IL-8 levels were significantly increased in moderate and severe cases and the levels of these cytokines were positively correlated with the severity of envenomation, as evaluated by clinical profile and plasma venom concentration. IL-10 levels were increased in severe and moderate cases and reduced in mild cases. The results reported in the present study suggest that the physiopathological manifestation of Ts envenomation may be mediated, at least in part, by cytokines, and that the early treatment after scorpion sting with drugs that inhibit cytokine production, such as glucocorticoids, may have a potential beneficial effect, ameliorating the severity of the clinical manifestations observed, particularly in severe and moderate cases.

Role of bradykinin in postprandial hypotension in humans.

A transient significant decrease in mean arterial blood pressure (MAP) from 107 +/- 3 to 98 +/- 3 mmHg (P < 0.05) was observed in elderly (59-69 years of age), healthy volunteers 25-30 min following ingestion of a test meal. In young volunteers (22-34 years of age), a postprandial decrease of MAP from 88 +/- 3 to 83 +/- 4 mmHg was also noted but it was not statistically significant. A 40% decrease in bradykinin (BK) content of circulatory high molecular weight kininogen had previously been observed in human subjects given the same test meal. We presently demonstrate by specific ELISA that the stable pentapeptide metabolite (1-5 BK) of BK increases from 2.5 +/- 1.0 to 11.0 +/- 2.5 pg/ml plasma (P < 0.05) in elderly volunteers and from 2.0 +/- 1.0 to 10.3 +/- 3.2 pg/ml plasma (P < 0.05) in young volunteers 3 h following food intake. This result suggests that ingestion of food stimulates BK release from kininogen in normal man. Postprandial splanchnic vasodilatation, demonstrated by a decrease of plasma half-life of intravenously administered indocyanine green (ICG), a marker of mesenteric blood flow to the liver, from 4.4 +/- 0.4 to 3.0 +/- 0.1 min (P < 0.05) in young volunteers and from 5.2 +/- 1.0 to 4.0 +/- 0.5 min (P < 0.05) in elderly volunteers, accompanied BK release. The participation of BK in this response was investigated in subjects given the BK-potentiating drug captopril prior to food intake. Postprandial decreases of ICG half-lives were not changed by this treatment in either young or elderly subjects, a result which may indicate that BK released following food intake plays no role in postprandial splanchnic vasodilatation in normal man.

Evidence for new spasmogenic substances released by trypsin from plasma of normotensive and hypertensive rats.

We have extended our previous study of increased levels of new spasmogenic substances released by trypsin from the plasma of chronic one-kidney, one-clip hypertensive rats by describing preparative procedures utilizing Sephadex G-25, CM-cellulose and C18 reverse-phase HPLC for their partial purification. We demonstrate the existence of at least 6 HPLC components, distinguishable from bradykinin, bradykinin homologues having Lys before Arg1 of bradykinin and T-kinin (Ile-Ser-bradykinin) on the basis of their chromatographic properties. Several of these spasmogenic substances induce a very slow contraction of the isolated guinea pig ileum when compared to bradykinin.

Hydrolysis of rat high molecular weight kininogen by purified rat urinary kallikrein: identification of bradykinin as the kinin formed.

We have previously reported that, although human urinary kallikrein, like glandular kallikreins for other species, releases lysyl-bradykinin from homologous and heterologous substrates, rat urinary kallikrein released a kinin which migrated like bradykinin in CM-cellulose chromatography and polyacrylamide gel electrophoresis (BBA677, 471, 1981). In the study we definitively established the nature of the kinin produced by rat urinary kallikrein by using purified enzyme and substrate, HPLC, radioimmunoassay and N-terminal analysis. Rat urinary kallikrein was purified to apparent homogeneity by a procedure which included affinity chromatography on aprotinin agarose. The kinin produced by rat urinary kallikrein acting on either pure rat high molecular weight kininogen or rat plasma or semipurified bovine and dog plasma was identified as bradykinin. This observation provides the evidence of species differences in the specificity of glandular kallikreins acting on kininogens.

[Cerebrospinal fluid in the diagnosis of cerebro-meningeal metastasis from malignant melanoma arising from giant congenital melanocytic nevus: case report]. / Líquido cefalorraquiano no diagnóstico de metástase cérebro-meníngea de melanoma maligno derivado de nevo melanocítico gigante congênito. Relato de caso.

We describe the case of a 28-year-old man with a giant congenital melanocytic nevus (GCMN) with malignant transformation to melanoma and metastasis on the central nervous system (CNS). We also make a summary of the pathological features from both lesions (GCMN and Melanoma), the occurrence of malignancy of GCMN, the organs more frequently involved with metastatic melanoma--with emphasis to involvement of CNS--just as the factors that cause malignant transformation of GCMN; the methods to diagnose metastases in CNS--emphasizing the importance of cerebrospinal fluid--and some therapeutical modalities for the metastatic melanoma in CNS.

Increased activity of plasma and tissue kallikreins, plasma kininase II and salivary kallikrein in pemphigus foliaceus (fogo selvagem).

BACKGROUND: Pemphigus foliaceus (PF) is an autoimmune blistering disease of unknown aetiology, which is endemic in Brazil. Although the pathogenesis of PF is still unknown, proteins of the contact system have been implicated. OBJECTIVES: As the components of the kinin system may interact with those of the contact system, in this study we evaluated the plasma levels of high-molecular-weight kininogen (HK) and low-molecular-weight kininogen (LK), and the activity of plasma kallikrein, tissue kallikrein and kininase II in plasma of patients with PF presenting with Nikolsky's sign. As kidneys and salivary glands are relevant sources of tissue kallikrein for plasma, we also evaluated urinary/salivary kallikrein and urinary kininase II activities. METHODS: Fifteen patients and 15 age- and sex-matched controls were studied. Kininogen levels were determined by enzyme-linked immunosorbent assay, and the activities of kallikreins and kininase II were determined using selective chromogenic substrates. RESULTS: Compared with controls, plasma HK levels were decreased (P = 0.031), whereas the activities of plasma kallikrein, tissue kallikrein and kininase II in plasma, and the activity of salivary kallikrein, were increased in patients (P < 0.001 for each comparison). Plasma levels of LK and the activities of urinary kallikrein and urinary kininase II were not significantly different from controls. CONCLUSIONS: Diminished levels of HK associated with increased activities of plasma kallikrein and kininase II indicate that the kinin system is activated at the systemic level in PF. As active plasma kallikreins may act on some proteins of the contact system, it is possible that the enzyme may contribute to blister formation. The further observation of an increased tissue kallikrein activity at the systemic and saliva levels may be interpreted as a systemic reflex of skin inflammation. Whether the activation of the kinin system is a cause or a consequence of blister formation needs further clarification.

Susceptibility of a peptide derived from bradykinin to hydrolysis by brain endo-oligopeptidases and pancreatic proteinases.

The property of brain endopeptidases of attacking small biologically active polypeptides but not denatured proteins led us to compare them with pancreatic proteolytic enzymes with respect to hydrolysis of a synthetic peptide derived from bradykinin (Gly-Gly-Gly-Arg-bradykinin), free, bound to Affi-Gel 10, or bound to succinylated polylysine of 3,000 and 180,000 daltons, respectively. The data show that brain endopeptidases A and B only hydrolyze bradykinin in its free form, whereas trypsin, chymotrypsin, and carboxypeptidase B hydrolyze the polypeptide both free and covalently bound to a high molecular weight carrier. These results suggest that brain endopeptidases selectively hydrolyze low molecular weight polypeptides.

Kininogen changes in the alloxan-diabetic rat.

Ten-day alloxan diabetic rats showed substantial increases in plasma T-kininogen, high molecular and low molecular weight kininogens, as well as significant decreases in urinary levels of kallikrein. Changes in T-kininogen were also observed following turpentine inflammatory treatment. In the diabetic rat, inflammatory responses may contribute to T-kininogen increases.

Kininogen-kallikrein-kinin system in plasma and saliva of patients with Sjögren's syndrome.

OBJECTIVE: To evaluate variables of the kininogen-kallikrein-kinin system (KKKS) simultaneously in plasma and saliva of patients with Sjögren's syndrome (SS). METHODS: We studied a group of 20 female patients with SS aged 37-75 years, 7 with primary SS (SS1) and 13 with SS secondary to rheumatoid arthritis (SS2), and 20 healthy individuals. Total kininogen and high and low molecular weight kininogen (HKg and LKg, respectively) levels were evaluated by ELISA. The activity of plasma and tissue kallikreins was determined by enzyme activity on selective chromogenic substrates. RESULTS: The plasma levels of total kininogen, HKg, and LKg, and the activity of plasma kallikrein observed in patients were not significantly different from controls. The tissue kallikrein-like activity in plasma and the active tissue kallikrein in saliva were significantly increased in patients with SS, whereas the total salivary tissue kallikrein activity in patients was not significantly different from controls. The concentration of protein in the saliva of patients was significantly increased, and a positive correlation between salivary protein levels and the active tissue kallikrein was observed. CONCLUSION: Comparisons between the total and the active tissue kallikrein in saliva of patients with SS showed that most of the tissue kallikrein was in its active form. In addition, we observed a concomitant increase of the tissue kallikrein-like activity in plasma. These results suggest increased activation of the KKKS in plasma and saliva of patients with SS.