Dynamics of hollow atom formation in intense x-ray pulses probed by partial covariance mapping.

When exposed to ultraintense x-radiation sources such as free electron lasers (FELs) the innermost electronic shell can efficiently be emptied, creating a transient hollow atom or molecule. Understanding the femtosecond dynamics of such systems is fundamental to achieving atomic resolution in flash diffraction imaging of noncrystallized complex biological samples. We demonstrate the capacity of a correlation method called "partial covariance mapping" to probe the electron dynamics of neon atoms exposed to intense 8 fs pulses of 1062 eV photons. A complete picture of ionization processes competing in hollow atom formation and decay is visualized with unprecedented ease and the map reveals hitherto unobserved nonlinear sequences of photoionization and Auger events. The technique is particularly well suited to the high counting rate inherent in FEL experiments.

SP-40,40, a newly identified normal human serum protein found in the SC5b-9 complex of complement and in the immune deposits in glomerulonephritis.

We report herein the isolation and initial characterization of a novel protein, termed SP-40,40, which is present at moderate levels (35-105 micrograms/ml) in normal human serum. SP-40,40 is deposited in the renal glomeruli of patients with glomerulonephritis but is not found in normal glomeruli. The protein is a heterodimeric structure of relative molecular mass 80 kD, both chains of which are of a similar size (40 kD). The amino-terminal sequences of both chains are unrelated to one another and possess no significant homology to any known protein sequence. The tissue distribution of SP-40,40 closely resembles that of the terminal complement components and its physicochemical properties are similar to, but distinct from, those of the S protein of complement. We have identified SP-40,40 in the SC5b-9 complex of complement and have demonstrated incorporation of labeled SP-40,40 into this complex. These data suggest that SP-40,40 is an additional component of SC5b-9.

Human seminal clusterin (SP-40,40). Isolation and characterization.

Molecular cloning of the human complement inhibitor SP-40,40, has revealed strong homology to a major rat and ram Sertoli cell product, sulfated glycoprotein-2, known also as clusterin. This study reports the purification and characterization of human seminal clusterin. Two-dimensional gel electrophoresis revealed charge differences between clusterin purified from semen and the serum-derived material. Both preparations demonstrate comparable hemagglutination (clustering) activity and inhibition of C5b-6 initiated hemolysis. The average clusterin concentration in normal seminal plasma is considerably higher than that found in serum. Mean seminal plasma clusterin concentrations were significantly lower in azoospermia caused by obstruction or seminiferous tubule failure than with oligospermia or normospermia. Only men with vasal agenesis had undetectable seminal clusterin, suggesting that some of the seminal clusterin is produced by the seminal vesicles. Immunofluorescence of human spermatozoa revealed that clusterin was detected on 10% of spermatozoa, predominantly those that were immature or had abnormal morphology. A pilot study of 25 patients suggests that seminal clusterin concentration, together with sperm motility and morphology, is correlated with the fertilization rate in vitro. The function of seminal clusterin is unknown. Its extensive distribution in the male genital tract and its high concentration in seminal plasma suggests an important role in male fertility.

Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease).

INTRODUCTION: Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology. SUBJECTS: Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded. RESULTS: Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls. CONCLUSION: We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.

Cutaneous necrosis from calcific uremic arteriolopathy.

Calcific uremic arteriolopathy (calciphylaxis) is an uncommon complication of chronic renal failure that is associated with high morbidity and mortality. We report 16 patients (13 female) who presented between 1985 and 1996. All patients developed painful livido reticularis that progressed to cutaneous necrosis and ulceration (11 cases on the proximal extremities and five cases on the distal extremities). Two patients with predominately distal leg disease survived; the cause of death in the other 14 patients was sepsis (six patients), withdrawal from dialysis (three), cardiac arrest (three), and gastrointestinal hemorrhage (two). Mesenteric ischemia from intestinal vascular calcification occurred in two cases. Clinical factors identified included the use of warfarin therapy in seven cases and significant weight loss (>10% body weight) in seven cases in the 6 months preceding the development of calcific uremic arteriolopathy. Skin pathology was studied in 12 cases, with all showing calcific panniculitis and small vessel calcification. Electron microscopic spectral analysis of the mineral content of the calcific lesions in the subcutaneous tissue showed only calcium and phosphorous. In two cases, substitution of low molecular weight heparin for warfarin therapy resulted in clinical improvement. Current theories of pathogenesis and treatment are reviewed. This study confirms the high morbidity and mortality of calcific uremic arteriolopathy producing ischemic tissue necrosis while drawing attention to significant weight loss and warfarin therapy as risk factors for the development of ischemic tissue necrosis. Hyperbaric oxygen therapy warrants further study.

Abnormal distribution of retinal clusterin in retinitis pigmentosa.

Increased expression of clusterin mRNA is associated with neurodegenerative states, including retinas affected by retinitis pigmentosa (RP). We have investigated the distribution of immunoreactive clusterin in normal and RP-affected retinas. Reactivity at the inner limiting membrane, plexiform layers, and photoreceptors in normal retina accords well with clusterin's postulated role as a membrane protective agent. In RP-affected retina the organized distribution is lost and overall reactivity appears decreased. The changes in this case may reflect increased turnover or removal of clusterin, perhaps via interaction with components of the immune system.

Comparison of the prophylactic effects of 2-deoxycytidine and prednisolone for high-dose intravenous cytarabine-induced keratitis.

In a double-masked, randomized fashion, 11 patients with hematologic malignancies received 13 courses of high-dose cytarabine therapy, intravenously (3 g/m2 every 12 hours for five to six days). Each patient received topical prednisolone phosphate 1% in one eye and 2-deoxycytidine 100 microM in the other eye every six hours. Topical therapy was initiated 12 hours before the first cytarabine dose and continued for up to ten days (until four to five days after completion of cytarabine therapy). Slit-lamp biomicroscopy was performed before therapy and then weekly for one month. 2-Deoxycytidine was equally as effective as the topical corticosteroid therapy in reducing photophobia and pain, microcysts, and punctate epithelial erosions, and each treatment gave results significantly better when compared historically to placebo-treated eyes.

Chronic Propionibacterium endophthalmitis after extracapsular cataract extraction and intraocular lens implantation.

We studied six cases of chronic, indolent intraocular inflammation that occurred after extracapsular cataract extraction and posterior chamber intraocular lens implantation. The inflammation was characterized by a delayed onset, and in three cases had the clinical appearance of a granulomatous iridocyclitis. Cultures of intraocular specimens obtained from six eyes yielded Propionibacterium; five yielded P. acnes. Pleomorphic gram-positive bacilli consistent with Propionibacterium were identified in cytologic or histopathologic studies in four of the six culture-positive cases. After surgical and medical therapy, the inflammation resolved. Postoperative Propionibacterium endophthalmitis may masquerade as a chronic iridocyclitis.

Identification of the components of glomerular immune deposits using monoclonal antibodies.

Monoclonal antibodies have been raised against components of glomerular immune deposits in experimental glomerulonephritis and idiopathic human glomerulonephritis. An accelerated model of chronic serum sickness in the rat using cationized human serum albumin was employed to obtain renal tissue with capillary loop and mesangial immune deposits. Mice were immunized with isolated rat glomeruli or a preparation of glomerular basement membrane and mouse spleen cells fused with myeloma cells. Anti-human serum albumin monoclonal antibodies were produced from all technically successful fusions irrespective of the size of the deposits in the immunizing tissue or whether whole glomeruli or glomerular basement membrane were used for immunization. Monoclonal antibodies were then produced following immunization with tissue from postmortem human kidneys with idiopathic membranous and mesangiocapillary glomerulonephritis. Sixteen monoclonal antibodies, apparently reactive with glomerular immune deposits, were cloned; most of these were reactive with components of the complement system including a previously undescribed complement-related protein. These studies demonstrate that monoclonal antibody technology may be useful in determining the identity of antigen and non-antigen components of glomerular immune deposits.

Localisation of clusterin in normal human sperm by immunogold electron microscopy.

In the human male reproductive tract, two forms of clusterin have been detected: the conventional heterodimeric form and a novel acrosomal form. On human sperm the novel form of clusterin is present in the acrosomal region of acrosome intact sperm only. The aim of this study was to determine the site of localisation of the acrosomal form of clusterin using immunogold electron microscopy on normal human sperm. Using the E5 anticlusterin mAb and a preembedding technique, acrosomal clusterin was localised in the acrosomal contents. Immunogold particles were detected on ethanol fixed spermatozoa that were subjected to Triton X-100 permeabilisation treatment. These sperm had lost their plasmalemma and outer acrosomal membrane. Specific immunogold labeling was present over the surface mainly of the acrosomal contents exposed by the loss of the plasma-lemma and outer acrosomal membrane. Immunogold particles were also detected in the equatorial segment of the sperm. These data confirm that the acrosomal form of clusterin is associated with the contents of the acrosome.

Localization of terminal complement components S-protein and SP-40,40 in renal biopsies.

The terminal complement complex has been implicated in the development of glomerular injury in both experimental and, indirectly, in human glomerulonephritis. Recent data suggests that the terminal complement complex in human glomerulonephritis may be in the cytolytically inactive SC5b-9 form which also contains S-protein and a recently identified protein, SP-40,40. In this study renal biopsies were examined by immunofluorescence to determine the incidence and inter-relation of deposition of the SC5b-9 components C6, C9, S-protein and SP-40,40. All components of SC5b-9 were found in arteries and arterioles, along the tubular basement membrane and in areas of glomerulosclerosis in all biopsies. This deposition was sometimes associated with C3 but never immunoglobulin deposition and correlated with the degree of renal injury. In addition, in biopsies with glomerular deposition of immunoglobulin and C3, the SC5b-9, components co-localized with the immune deposits. Glomeruli without immune deposits or glomerulosclerosis contained none of the SC5b-9 components. The incidence and pattern of distribution of SP-40,40 was similar to that of S-protein, C6 and C9 in all of cases. These data confirm that the terminal complement complex in the kidney is, at least partly, in the SC5b-9 form both in the specific immune glomerular deposition and in the "non-specific" deposition in areas of renal injury. SP-40,40 is also found in the SC5b-9 complex in all forms of renal disease.

Serum sickness nephropathy in the rat using cationized albumin: effect of preimmunization and antigen dose.

A model of glomerulonephritis induced in preimmunized rats with cationic albumin is described. Extensive glomerular immune complex formation and a severe nephrotic syndrome occurred within 5 days of commencement of daily intravenous injections. Severity of disease was markedly influenced by the degree of preimmunization and, to a lesser extent, by the dose of cationic albumin administered. Immune deposits, although initially confined along the capillary loops, were seen at all sites in the glomerulus. This study confirms that, in rats, the use of cationic antigens accelerates the development of 'serum sickness' nephropathy but preimmunization is necessary to produce significant disease.

Idiopathic membranous glomerulonephritis: long-term follow-up in 139 cases.

The clinical course of 139 patients (77 male, 62 female) with idiopathic membranous glomerulonephritis is reviewed. The median duration of follow-up was 52 months; 45% and 25% were followed for more than 5 and 10 years respectively. The median age at presentation was 36. Fifty-four percent of patients had the nephrotic syndrome at presentation. Half of the patients were treated at some stage with cyclophosphamide or corticosteroids. During the course of follow-up some deterioration in renal function occurred in only 20% of patients. The patients who suffered deterioration in renal function were mainly male and had significantly worse renal function and a higher incidence of the nephrotic syndrome than the other patients at presentation. Only 7 male patients (5%) developed terminal renal failure during follow-up and one female presented in terminal renal failure. Survival was 88% and 81% at 5 and 10 years. The median predicted (or actual) time for development of terminal renal failure in patients with progressive deterioration was 7.3 years. These data are in accord with other recently published series which have described a relatively benign prognosis for idiopathic membranous glomerulonephritis.

Randomized controlled trial of cyclophosphamide, warfarin and dipyridamole in idiopathic membranous glomerulonephritis.

40 patients with idiopathic membranous glomerulonephritis were randomized to receive either no treatment or a regime of cyclophosphamide for 6 months, and warfarin and dipyridamole for two years. During the two years of the trial there was no significant deterioration in renal function in either group. A significantly greater improvement in urinary protein excretion was, however, observed at all time points in the treatment group. Plasma albumin was also significantly higher in the treatment group at 18 and 24 months. As progressive deterioration in renal function in membranous glomerulonephritis is associated with persistent heavy proteinuria these results suggest a beneficial effect of treatment.

Antineutrophil cytoplasm antibody (ANCA) associated vasculitis.

In 1982 we first reported the presence of antineutrophil cytoplasm antibodies (ANCA) in 8 patients with systemic vasculitis and segmental necrotizing glomerulonephritis. The results of long-term follow-up are described. Screening of 7,500 serum samples revealed positive ANCA in 9 additional patients with vasculitis. Eighty-eight other patients with vasculitis were ANCA negative, including 7 with microscopic polyarteritis nodosa (MPAN) and 8 with Wegener's granulomatosis (WG). Conversely, ANCA were never detected in the absence of vasculitis. Fourteen patients presenting with glomerulonephritis and ANCA were followed for a median of 6.3 years. Eleven patients had MPAN and 3 WG. Remissions were obtained with immunosuppressive therapy in all patients. Clinical relapse was associated with the reappearance of ANCA. Five-year survival was 89% and 5-year dialysis free survival was 77%. ANCA are specific markers for a sub-group of patients with vasculitis and are sensitive markers of disease activity. Glomerulonephritis associated with ANCA positive vasculitis has a favorable outcome with immunosuppressive therapy.

The effect of immunosuppression on vascularised allografts. A preliminary report.

Five vascularised allografts of the knee joint were performed in dogs immunosuppressed with cyclosporin A and azathioprine. Three survived with normal function for 3 to 4 months after operation. One of the unsuccessful grafts had a failed vascular anastomosis, the other an inadequate blood level of cyclosporin A. All three successful grafts healed well. In two, bone scans, radiographs and biopsies were indistinguishable from successful autografts; in the third the blood supply to the graft failed despite patent anastomoses but the graft healed well with good function. All three grafts were rejected within 2 to 3 weeks of withdrawal of cyclosporin A and azathioprine. In non-immunosuppressed dogs, allografts of the knee, both vascularised and non-vascularised, were rejected within a few days of operation. In two non-vascularised allografts, administration of cyclosporin and azathioprine had no apparent effect on the rate of rejection of the graft.

Emerging photon technologies for probing ultrafast molecular dynamics.

The understanding of physical and chemical changes at an atomic spatial scale and on the time scale of atomic motion is essential for a broad range of scientific fields. A new class of femtosecond, intense, short wavelength lasers, the free electron lasers, has opened up new opportunities to investigate dynamics in many areas of science. For chemical dynamics to advance however, a rigorous, quantitative understanding of dynamical effects due to intense X-ray exposure is also required. We illustrate this point by reporting here an experimental and theoretical investigation of the interaction of C(60) molecules with intense X-ray pulses, in the multiphoton regime. We also describe the potential of new available instrumentation and explore their potential impact in physical, chemical and biological sciences when they are coupled with emerging photon technologies.

Femtosecond X-ray-induced explosion of C60 at extreme intensity.

Understanding molecular femtosecond dynamics under intense X-ray exposure is critical to progress in biomolecular imaging and matter under extreme conditions. Imaging viruses and proteins at an atomic spatial scale and on the time scale of atomic motion requires rigorous, quantitative understanding of dynamical effects of intense X-ray exposure. Here we present an experimental and theoretical study of C60 molecules interacting with intense X-ray pulses from a free-electron laser, revealing the influence of processes not previously reported. Our work illustrates the successful use of classical mechanics to describe all moving particles in C60, an approach that scales well to larger systems, for example, biomolecules. Comparisons of the model with experimental data on C60 ion fragmentation show excellent agreement under a variety of laser conditions. The results indicate that this modelling is applicable for X-ray interactions with any extended system, even at higher X-ray dose rates expected with future light sources.