Experimental murine IgA nephropathy following passive administration of vomitoxin-induced IgA monoclonal antibodies

Oral exposure of mice to vomitoxin (VT) induces elevated levels of serum IgA, circulating IgA immune complexes (IgA-IC), mesangial IgA deposition and haematuria, which all mimic the clinical signs of human IgA nephropathy (IgAN). To further assess the effects of VT-induced IgA in the murine model, B6C3F1 and BALB/C mice were injected intraperitoneally with affinity-purified monoclonal IgA derived from Peyer's patch hybridomas of VT-exposed mice. In B6C3F1 mice, serum IgA, IgM and IgA-IC levels were increased two- to fivefold in treatment groups after 4 and 6 wk compared with controls, whereas increases in serum IgG as high as 18-fold were observed. Urinary erythrocyte counts were also significantly elevated in treatment groups after 2, 4 and 6 wk compared with controls. Concurrent increases in IgA and IgG complexes containing casein, the dietary protein source, occurred in treatment mice. Mesangial IgA, IgG, IgM and C3 deposition were significantly increased in all treatment mice after 6 wk. Electron-dense deposits occurred in the glomeruli of IgA-injected mice after 6 wk. All the above parameters were similarly affected in BALB/C mice. Injection of IgA-secreting hybridoma cells into BALB/C mice increased serum IgA, IgA-IC and IgG levels as well as elevated mesangial IgA, IgG and C3 deposition and haematuria after 2-3 weeks compared with controls. In total, these data indicate that passive administration of VT-induced IgAs can induce the hallmarks of IgA nephropathy. Casein, an antigen found in the diet used for these mice, appeared to form IC with IgA or IgG and these IC may participate in the pathogenesis of this nephropathy.     

Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state

The heterogeneity of follicle stimulating hormone (FSH) and luteinizing hormone (LH) was investigated in five women aged 29.4 +/- 3.2 years (mean +/- SD) throughout their menstrual cycles and in five post-menopausal women aged 53.8 +/- 5.6 years. Chromatofocusing (pH range 7-4) revealed menstrual cycle stage- and postmenopausal-related differences in the serum gonadotrophin charge. There were differences in the proportion of FSH with an isoelectric point (pl) > 4.3 across phases of the menstrual cycle (P = 0.019): midcycle (MC) 50%; early to mid-follicular (EMF) 36%; late follicular (LF) 37%, luteal (L) 29% and following the menopause (PM) 17%. There was no significant difference in the proportion of LH with pl > 6.55 between midcycle (53%) and EMF, LF or L phases (36, 43 and 32% respectively); although all were greater than that found in the menopause (13%). Concanavalin A chromatography revealed less (P < 0.005) complex FSH and LH glycoforms at midcycle (63 and 13%) than in the EMF, LF and L phases (90 and 18; 90 and 20 and 93 and 24% respectively). Menopausal gonadotrophins were least complex (FSH 34%, LH 4%). There was a direct relationship between serum FSH and FSH pl/complexity, and less acidic FSH was associated with reduced FSH complexity. Increased oestradiol was associated with basic FSH isoforms during the menstrual cycle and reduced follicular phase FSH complexity. We conclude that changes in gonadotrophin glycoforms occur through the menstrual cycle which are related to changes in the prevailing steroid environment. Following the menopause oestrogenic loss resulted in acidic, relatively simple glycoforms.     

Characterization of the major proteins of bovine seminal fluid by two-dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (M(r)) between 6 and 100 kDa. isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a M(r) of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its M(r) to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their M(r) nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content.     

Ambiguous and obscure problems of liver resection

Earlier work has suggested that up to 25% of children undergoing tonsillectomy because of recurrent sore throats are relatively deficient in IgA immunoglobulin and that they do less well after the operation than 'normal' children. Measurements of serum immunoglobulin levels were carried out in 96 children undergoing tonsillectomy for recurrent sore throats. Levels of IgA, IgM and IgG were found to be similar to those in healthy children, 7-29% of those studied had 'low' IgA serum levels. There was a significant relationship between serum IgA levels and the age of the child. No relationship could be established between IgA levels and pre- or postoperative clinicaal state of the children. Low IgA serum levels are probably the result of delayed immunological maturation rather than a true immunodeficient state. IgA estimations, therefore, have no bearing on selection for operation or the prognosis after operation.     

Clinical studies of the prognosis in cases of chance proteinuria and/or hematuria]

A total of 174 cases that consulted due to chance proteinuria and/or hematuria (CPH) were studied as to its clinical course, in particular patients' prognosis. They were selected from 311 patients on whom renal biopsy was performed from December, 1975 to December, 1985 in our institute. Furthermore, IgA nephropathy which occupied the major part of the CPH group was also studied as a prognostic factor. The CPH group showed 81% of disease stabilizing rate in 10 years' follow-up. In various data such as chemical analysis of blood and urine, immunoglobulin levels, and renal function at the time of biopsy, daily urinary protein excretion (greater than 1 g/day) statistically showed a significant correlation to deterioration of the renal function during the follow up. However, hematuria was not found correlated. Of CPH group, 48% was diagnosed to be with IgA nephropathy. The patients with IgA nephropathy with CPH, comparing with the cases without CPH, were younger and had better renal function and milder change of renal mesangial proliferation. The 10 years-disease stabilizing rates of the disease were 81% in CPH and 63% in non CHP group. In conclusion, prognostic factors affecting renal function in the CPH group was found to be daily urinary protein excretion and, if diagnosed as IgA nephropathy by biopsy, pathological changes were also shown to be prognostic factors. Therefore, CPH patients having proteinuria over 1 g/day must be examined by renal biopsy and when IgA nephropathy is diagnosed, long time follow-up is necessary and re-biopsy for examination of pathological change during the interval is recommended.     

IgA levels and carrier rate of pathogenic bacteria in 27 children previously tonsillectomized

The object of the present paper is to present laboratory and clinical data on 27 children of ages between 6 and 11 years, who in connection with tonsillectomy 2 1/2 years earlier had been found to have low serum and saliva IgA levels, low serum IgE levels, and a considerable lack of IgA and IgE plasma cells in the excised tonsils; correlation between deficiency in IgA and culture of pathogenic bacteria from the tonsils was significant. From a clinical point of view, 22 of the children had benefit of the tonsillectomy and had no longer a tendency towards a development of recurrent infections. The remaining 5 patients continued to complain of recurring respiratory infections; in addition, levels of serum and saliva IgA were low. Furthermore, 4 or these 5 children harboured pathogenic bacteria in their throats. Many of the 27 patients still had low serum IgA and IgE levels as compared with levels in healthy, age-related controls; in 3 patients, however, the IgE levels in serum had risen considerably parallel with the development of atopic diseases. Saliva IgA was rather constant after tonsillectomy as compared with the preoperative levels, though it had risen in some of the children. As regards serum IgG and IgM, these immunoglobulins had decreased significantly, and the question is raised, whether it might had been due to the tonsillectomy, either by the removal of chronically infected organs or by the removal of important immunological tissue.     

The intra- and interobserver variability of impedance cardiography in patients at rest and during exercise

In the present work we studied the levels of activities of dipeptidyl-peptidase I (or cathepsin C, DPP-I) and dipeptidyl-peptidase II (DPP-II) and examined their isoelectric focusing profiles in matched pairs of human squamous cell lung carcinoma (SQCLC) and the lung from surgically treated patients (n = 33). The mean specific activities of DPP-I and DPP-II were higher in SQCLC (Stages I and II) than in the lung, but only the activity of DPP-II in Stage I SQCLC was significantly higher compared to the lung. The activities of both enzymes were higher in the tumor than in the lung in 10 of 20 Stage I SQCLC patients, but only in 3 of 13 Stage II SQCLC patients. The specific activities of DPP-I and DPP-II in the lungs showed a good correlation while the correlation of both enzyme activities in SQCLCs was poor. We observed only a small and mutually comparable activation of DPP-I in extracts from SQCLCs and from the lungs by dithiothreitol. The isoelectric focusing profile of several DPP-II forms in SQCLCs and the lungs was similar and the single major DPP-II isoform revealed in the tumors and lungs showed a pIapp of 5.3-5.2. The isoelectric focusing profile of DPP-I showed multiple enzyme forms in SQCLCs (pIapp 6.3-4.5) as well as in the lungs (pIapp 6.4-4.8). In SQCLCs, as well as in the lungs, the activities of the DPP-I forms with pIapp values < or = 5.6 were shifted by neuraminidase treatment to the site of the major DPP-I isoform with pIapp of about 6.0 and the zymograms then showed an another DPP-I with pIapp of 5.7, which was less discernible in the lung. In some patients, the DPP-I forms with pIapp values < or = 5.6 from SQCLC retained a greater percentage of activity distribution than did the DPP-I pIapp-counterparts from the lung.     

Consequences of the latest Euratom directive 96/29 ("basic standard") regarding radiotherapy]

BACKGROUND AND OBJECTIVES: To determine the prevalence rates of serological reactivity of Haemophilus (H.) ducreyi, Treponema pallidum, and herpes simplex virus type 2 (HSV-2) antibodies among female sex workers (FSWs) and their association with human immunodeficiency virus (HIV) antibody status. STUDY DESIGN: Cross-sectional, standard serological assays were used for syphilis, HSV-2 and HIV; a modified enzyme-linked immunosorbent assay (ELISA) was used to detect specific anti-H. ducreyi immunoglobulin (Ig) G and IgA antibodies. RESULTS: Seroprevalence rates were 86% for anti-H. ducreyi IgG and 69% for anti-H. ducreyi IgA; 4% for rapid plasma reagin (RPR) and Treponema palladium hemagglutination assay (TPHA) confirmed syphilis; 59% for HSV-2; 12% for HIV-1 and 2% for HIV-2. Lower-class FSWs were significantly more likely than upper-class FSWs to be H. ducreyi seropositive (IgG: OR = 42.7; IgA: OR = 7.6) and have current or past syphilis infection (RPR: OR = 3.5; RPR and TPHA: OR = 4.5). The presence of syphilis increased significantly with older age (P-trend < 0.001). Non-Nigerian FSWs had significantly higher reactivity to chancroid (IgG: OR = 3.5; IgA: OR = 1.8) and borderline reactivity to syphilis (RPR: OR = 1.6; TPHA: OR = 2.0). A history of sex with non-Nigerian Africans was significantly associated with chancroid reactivity and borderline significant with syphilis serostatus. H. ducreyi seropositivity was significantly more likely in FSWs with HSV-2 (OR = 2.4) and syphilis (OR = 5.6). Chancroid and HSV-2 antibodies were also more common in HIV-infected FSWs. CONCLUSION: The prevalence of H. ducreyi antibodies is the highest rate that has been reported. Our findings underscore the importance of an effective program to control GUDs as part of the strategy to prevent the potentially explosive spread of HIV in Nigeria.     

Thin GBM nephropathy: premature glomerular obsolescence is associated with hypertension and late onset renal failure

Thin glomerular basement membrane (GBM) nephropathy, also called familial benign hematuria, is characterized by chronic hematuria and uniform thinning of the lamina densa of the glomerular basement membrane. It generally holds an excellent renal prognosis. Alport syndrome in early stages can also show attenuation of the GBM; conversely, renal insufficiency has been reported in familial benign hematuria. To discern early Alport syndrome from thin GBM nephropathy, we carried out a prospective epidemiological study in which 19 normotensive and non-azotemic adult patients with chronic microscopic (18 of 19) and macroscopic (1 of 19) hematuria and biopsy-proven thin GBM nephropathy were followed for a median of 12 years (range 9 to 15 years). Renal biopsies of thin GBM patients at entry showed an increased incidence of focal global glomerulosclerosis when compared to disease controls as IgA nephropathy (P = 0.047) and normal renal tissue (P = 0.0075). All renal biopsies showed the presence of the Goodpasture antigen when tested immunohistochemically. Presence of Alport syndrome was excluded clinically as none of the patients had complaints of hearing loss or abnormalities by audiography and ophthalmology. At the end of follow-up, the incidence of hypertension in thin GBM nephropathy (35%) exceeded that of healthy clinical controls (P = 0.048), and one hypertensive patient developed mild renal failure. In the normotensive patients, the glomerular filtration rate at follow-up as measured by inulin clearance was reduced in three out of seven; these were over 50 years of age. Although no family members were known to have renal disease at inclusion, within four families six elderly first degree relatives had developed unexplained renal insufficiency at the end of follow-up. Thus, thin GBM nephropathy predisposes to premature glomerular obsolescence, leading in time to increased incidences of hypertension and late onset renal insufficiency.     

Glomerular permeability: in vivo tracer studies with polyanionic and polycationic ferritins

The influence of molecular charge on glomerular permeability to the globular ferritin molecule in vivo was investigated. Mice (Charles River CD strain) and rats (Munich-Wistar strain) were injected intravenously either with native anionic ferritin or various cationized derivatives with different isoelectric points (pI) and the kidneys were examined by electron microscopy. Native anionic ferritin was almost completely restricted from entry into the glomerular filter at the level of the endothelium and subendothelial layer of the glomerular basement membrane (GBM). Cationized derivatives penetrated the filter in increasing amounts depending on the pI of the tracer. Regardless of charge, all molecules that filtered through the lamina densa of the GBM and reached the subepithelial layer were completely restricted from entry into the urinary space at the level of filtration slits and appeared in phagosomes present in podocytes. Reduction of arterial pressure of cessation of renal blood flow did not influence the movement of ferritin molecules into the GBM. The results are consonant with physiological studies indicating charge dependent restriction of polyanion transport by the mammalian glomerulus. These tracer studies, in conjunction with cytochemical and biochemical evidence for the presence of polyanionic glycoproteins in the glomerular filter, suggest that glomerular restriction of plasma proteins occurs in part by a process similar to that which exludes negatively charged macromolecules in polyanionic gel systems.     

Membranoproliferative glomerulonephritis induced by portosystemic shunt surgery for non-cirrhotic portal hypertension

The liver and spleen both have important phagocytic functions and contain monocytes/macrophages which clear immune complexes. We describe here three patients who presented proteinuria and hematuria 7 to 13 years after portosystemic shunt surgery, which diverted portal venous blood to the systemic circulation. They had hematemesis and/or melena and underwent mesocaval shunt surgery and splenectomy in childhood because of non-cirrhotic portal hypertension with esophageal varices. Renal biopsy specimens revealed findings characteristic of membranoproliferative glomerulonephritis (MPGN) type I. Immunohistologically, these three cases were accompanied by a distinct IgA deposition along with a marked C3 deposition. The IgA observed in these three cases contained not only IgA1 but also IgA2, which is the predominant form of mucosal IgA. On the other hand, of 20 patients with idiopathic MPGN type I with IgA deposition (n = 20), only two were positive for IgA2, and the distribution was focal and segmental. Our study shows that MPGN type I may have developed secondary to portosystemic shunt. This secondary form of MPGN type I may be caused by a reduced clearance of immune complexes in the liver and their deposition in the glomerulus, since a portosystemic shunt routes portal venous blood from the intestinal tract directly to the systemic circulation.     

Long-term prognosis for tonsillectomy patients with IgA nephropathy

A retrospective study of 85 patients with IgA nephropathy was undertaken to determine the long-term effect of tonsillectomy. Forty-three patients (24 males and 19 females) had received tonsillectomies (Group A) and 42 patients (17 males and 25 females) had not (Group B). These patients had been followed up for more than 5 years after renal biopsy. The average age at the initial renal biopsy was 25.72 years in Group A, and 33.16 years old in Group B. The average period of renal biopsy to tonsillectomy in Group A was 10.47 months. The average follow-up period was 8 years and 9 months in both groups. At the beginning of treatment, the two groups were well matched in terms of creatinine clearance, urinalysis, and blood pressure. Six patients in Group A and eight patients in Group B were treated with steroids. The glomerular injury detected at the renal biopsy was more extensive in Group A than in Group B. Renal function in the two groups was compared. The clinical remission rate in Group A was significantly higher than in group B (P<0.05). The stable renal function rate in Group A was significantly higher than in Group B (P<0.05). The renal survival rate was 97.7% in Group A and 83.3% in Group B, but there was no significant difference between the two groups. Histologically, the rate of remission of the minor lesion in Group A was significantly higher than in Group B (P < 0.05). Our results showed that tonsillectomy for IgA nephropathy was clinically of great value.     

Shaker potassium channel gating. II: Transitions in the activation pathway

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.     

Internal carbohydrate complexity of the oligosaccharide chains of recombinant human follicle stimulating hormone (Puregon, Org 32489): a comparison with Metrodin and Metrodin-HP

Glycoforms of recombinant human follicle stimulating hormone (rhFSH) (Org 32489, Puregon) were characterized using concanavalin A lectin affinity chromatography to reveal information about the internal carbohydrate complexity (extent of carbohydrate side-chain branching) of the preparations. The rhFSH glycoforms were measured by radioimmunoassay and a two-site immunoradiometric assay and compared with those in two urinary preparations (Metrodin and Metrodin-HP) used in assisted reproduction programmes and a urinary FSH international standard 70/45 (uFSH IS 70/45). Similar data were obtained with both assays; rhFSH had 6% complex internal carbohydrate structures compared with 22-27% for Metrodin, Metrodin-HP and uFSH. The proportion of simple carbohydrate structures was also different, with rhFSH having 18.5 compared with 4.5-9.3% for Metrodin, Metrodin-HP and uFSH. A linear relationship was observed between the percentage glycoforms with an isoelectric point (pl) < 4 and the log percentage simple forms (logarithmic regression; r = 0.93) indicating a direct relationship between carbohydrate complexity and charge heterogeneity. In summary, rhFSH contains fewer complex forms and an increased proportion of simple carbohydrate structures in comparison with Metrodin, Metrodin-HP and IS 70/45.     

Benign and malignant teratomas in children: analysis of 85 patients

Teratomas were observed in 85 infants and children. Fifty-eight (68%) were girls and 27 (32%) boys. Site of origin was sacrococcygeal in 55 patients (64.8%), mediastinal in ten (11.7%), gonadal in ten (11.7%), presacral in four (4.8%), retroperitoneal in three (3.5%), and cervical in three (3.5%). Sixty-seven (78.8%) teratomas were benign and 18 (21.1%) malignant. Malignant tumors were noted in 11 of 55 sacrococcygeal (20%), two of ten mediastinal (20%), three of eight ovarian (37.5%), and both testicular lesions. Cervical, retroperitoneal, and presacral tumors were benign. Age at diagnosis (greater than 1 month), presence of symptoms (urinary and colonic obstruction), and serum positive for alpha fetoprotein were indicators of malignancy in sacrococcygeal cases. Age was not a factor for teratomas at other sites. Tumor size, presence of calcification, and gross appearance (cystic or solid) did not relate to the tumor's benign or malignant nature. Three of four presacral tumors were associated with anorectal anomalies. Operative resection is the treatment of choice. Four deaths were related to operative hemorrhage. Four of five survivors with malignant teratoma received radiation and chemotherapy. A more aggressive role for combined adjunctive measures is suggested in cases of malignancy.     

Use of computerized tomography to evaluate bleeding after renal biopsy

Perirenal bleeding following biopsy was assessed in 25 consecutive cases using computerized axial tomography (CT) scanning. Perirenal hematomas were found in 15 patients (60%). In 8 patients the hematomas were moderate or large in size. There was no correlation between the clinical findings, fall in hemoglobin, presence of macroscopic hematuria and the finding of perirenal hematoma by CT scanning.     

The effect of tonsillectomy on the level of circulating immune complexes and urine changes in patients with glomerulonephritis

The influence of tonsillectomy on circulating immune complexes (C.I.C.) level, proteinuria and erythrocyturia was studied in 42 patients with chronic tonsillitis (Ch.T.) and urine abnormalities. The level of C.I.C. was examined by two methods: the 3.5% polyethyleneglycol (PEG) precipitation method and the 125I-C1q binding method. After tonsillectomy, bacteriological analysis of removes facial tonsilla was performed in 7 patients and morphological analysis in 11. Renal biopsy was done in 28 patients. The control group was consisted of 18 patients with Ch.T. without urine abnormalities. The presence of C.I.C. was established in 48% of patients with urine abnormalities using PEG method and in 33% with 125I-C1q binding method. Mean values of C.I.C. in patients with proteinuria or erythrocyturia were statistically higher than in the control group. After tonsillectomy, transitory increase of C.I.C. level was observed in 60% of patients, accompanied by augmentation in urine changes, especially proteinuria. During one year of observation, significant decrease in C.I.C. levels detected by PEG method, as well as in proteinuria and in erythrocyturia was found. In 10 patients urine abnormalities disappeared. No differences between both groups of patients were found in the results of bacteriological and morphological studies of removed tonsilla. However, the normalisation of urine changes was noticed in patients without hypertension and in whom renal disease did not exceed two years. Renal histology revealed mesangocapillary proliferative Gn in 14, mesangial proliferative Gn in 11, and focal/segmental glomerulosclerosis in 3 patients. In one patient with mesangial proliferative Gn complete retreat of urine changes was observed. We suggest that the presence of Ch.T. influences on the C.I.C. detectability in patients with chronic glomerulonephritis. The tonsillectomy can lead do the decrease of C.I.C. levels, as well as to the decrease of proteinuria and/or erythrocyturia. Serum C.I.C. examination seems to be helpful in qualifying patients with Ch.T. for tonsillectomy, in immunological monitoring after the operation and in later prognosis in case of chronic glomerulonephritis.     

Electrophoretic separation of recombinant tissue-type plasminogen activator glycoforms: validation issues for capillary isoelectric focusing methods

Attempts were made to validate a capillary isoelectric focusing (cIEF) method for a recombinant glycoprotein as an alternative technique to slab gel isoelectric focusing methods routinely used to monitor such charge heterogeneity. The cIEF method principally separates the charged glycoforms of recombinant tissue-type plasminogen activator (rt-PA) on the basis of their sialic acid content. Nine to ten distinct peaks were consistently resolved, with the profile dependent on the class of ampholyte used. The pI of rt-PA measured with synthetic pI standards was in the range pH 6.5-7.5 with the migration of the standards affected by the presence of the protein. The method showed an acceptable recovery of > 100% and had good sensitivity where 25 ng of protein could be resolved into constituent peaks. Recovery of both major peaks and total protein measured by peak areas was linear over a wide range from 50-1000 micrograms/mL. A detailed study showed that when a capillary had been used for some time, capillary age affected peak migration times and, to a lesser extent, resolution. Peak migration times were stable over a temperature range of 15-30 degrees C, and decreased predictably with increasing voltages (400-600 V/cm) and decreasing N,N,N',N'-tetramethylethylene diamine (TEMED) concentrations (0.4-1.5% v/v). Overall the data indicated that this methodology has the potential to be used in the commercial release of protein pharmaceuticals if variability resulting from capillary age and lot were resolved. Even in its present format the method equals the performance of slab gel IEF whilst offering significant improvements in ease of operation and in time and reagent use.     

Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using Dot-blot, ELISA and western-blot

Twenty-seven mycologically proven cases of paracoccidioidomycosis (PCM) were treated with itraconazole (100-200 mg/day in month 1 and 100 mg/day until month 6-8) and evaluated clinically and serologically, up to 3.5 years post-therapy, using Dot-blot and ELISA for measuring the titers of IgG, IgA and IgM anti-P.brasiliensis antibodies and Western-blot for determining IgG, IgA and IgM antibodies against the antigen components of the fungus. Before treatment, 81.5% (Dot-blot) and 84% (ELISA) of the patients presented elevated IgG anti-P.brasiliensis antibody titers which dropped slightly with treatment. On the other hand, the percentages of pre-treatment high-titered sera for IgA and IgM anti-P.brasiliensis were lower (51.9% and 51.8%: Dot-blot; 16.5 and 36%: ELISA, respectively) but the titers tended to become negative more frequently with treatment. Prior to treatment, the percentages of positivity for IgG, IgA and IgM anti-P.brasiliensis antibodies in Western-blot were 96%, 20.8% and 41.6%, respectively. Antigens with molecular weights varying from 16-78 kDa, from 21-76 kDa and from 27-78 kDa were reactive for IgG, IgA and IgM antibodies, respectively. The most frequently reactive antigenic components had molecular weights of 27, 33 and 43 kDa for IgG, and 70 for IgA and IgM antibodies. During the period of study, the patients responded well to treatment. The present data confirm the diversity and complexity of the humoral response in PCM, and the importance of utilizing different serological tests to detect IgG, IgA and IgM anti-P. brasiliensis antibodies.     

The role of liver transplantation in the subacute trauma patients

A 48-year-old man presented with painless left lateralizing macroscopic hematuria. Ureteroscopy revealed flocculent material in the left kidney and white linear streaks on some upper pole papillae; urine culture from the left renal pelvis was positive for tuberculosis, whereas voided urine cultures were negative. Renal tuberculosis should be included in the differential diagnosis of lateralizing hematuria, especially in the absence of an obvious cause for the bleeding. Direct culture of urine from the renal pelvis may have more sensitivity than culture of voided urine in this circumstance.