Proteolysis and partial dissolution of calcium oxalate: a comparative, morphological study of urinary crystals from black and white subjects

Crystal adherence to the renal epithelium is widely regarded as a probable mechanism of stone formation. Intracrystalline proteins may provide access to the core of urinary crystals and thereby facilitate the dismantling of these crystals after their attachment to and phagocytosis by renal epithelial cells. The present study investigated the role of proteolysis and limited dissolution of urinary calcium oxalate (CaOx) crystals in South Africas white and black populations with a view to understanding the remarkably low stone incidence in the black population compared with the whites. CaOx crystals were precipitated from filtered urine or ultrafiltered urine dosed with an intracrystalline protein, urinary prothrombin fragment 1 (UPTF1), isolated from white and black subjects. The crystals were fractured and subjected to proteolysis and/or limited dissolution before examination using field emission scanning electron microscopy (FESEM). FESEM data showed that CaOx crystals from white and black subjects were eroded by treatment with proteases. Cathepsin D caused the most significant crystal erosion, and more noticeable degradation of CaOx monohydrate (COM) crystals compared to CaOx dihydrate (COD). Limited dissolution studies showed the unique ultrastructures and fragmentation processes of COM and COD crystals. COM crystals appeared to be more susceptible to degradation and dissolution than CODs. Since COMs are predominant in blacks, compared with COD crystals from whites, it is speculated that the lower stone rate amongst South African blacks might be attributed partly to their more efficient destruction of retained COM crystals.     

Phenotypic and functional characteristics of intestinal intraepithelial lymphocytes during acute rejection of small intestinal allografts

Infiltration of a transplanted organ by host lymphoid cells is the hallmark of acute rejection. However, after intestinal transplantation, physiological lymphocyte migration may lead to host cell infiltration of the graft even in the absence of rejection. It is unclear whether this lymphocyte migration also involves the intraepithelial compartment of the graft or whether infiltration there is indicative of acute rejection. We demonstrate here that host cell infiltration of the intestinal mucosa occurs both during acute rejection of a small bowel allograft and, to a lesser extent, when rejection is prevented by immunosuppression with FK506. The infiltrating host cells consisted of CD3 ⁺ T cells with a predominant CD4^–CD8 ⁺ phenotype resembling intraepithelial lymphocytes (IELs). Functional studies showed that the nonspecific cytolytic activity of IELs was not affected by acute rejection or by immunosuppression with FK506. These findings indicate that host cell infiltration of the intestinal mucosa does not connote an ongoing acute rejection. Furthermore, the decreased mucosal barrier function during acute rejection of intestinal allgrafts is probably not due to impaired cytolytic activity of IELs. Received: 10 March 1997 Received after revision: 6 November 1997 Accepted: 19 November 1997     

Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals

OBJECTIVE: To compare the ultrastructure and protein content, particularly prothrombin fragment 1 and osteopontin, of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals precipitated from human urine, and their susceptibility to proteolysis, to try to clarify the role of intracrystalline proteins in urolithiasis, as differences between these types of crystal may determine whether calcium oxalate crystals nucleated in urine progress to stone formation. MATERIALS AND METHODS: Sodium dodecyl sulphate gel electrophoresis and Western blotting were used to analyse demineralized extracts of COM and/or COD crystals deposited from the same centrifuged and filtered urine (which contains abundant urinary proteins) by adjusting the calcium concentration to 2 and 7 mmol/L, respectively. Similar analyses were performed on COM and COD crystals deposited from ultrafiltered urine (which contains only proteins of < 10 kDa) and then incubated in centrifuged and filtered urine, as well as crystals generated in the presence of increasing concentrations of proteins derived from the organic matrix of urinary calcium oxalate crystals. Field-emission scanning electron microscopy was used to assess effects of proteinase K and cathepsin D on internal and superficial crystal structure. RESULTS: Osteopontin was undetectable in COM extracts, but clearly visible in COD. Prothrombin fragment 1 was abundant in COM, but present in COD in lesser amounts than osteopontin. The selectivity was also the same with crystals from ultrafiltered urine that were incubated in centrifuged and filtered urine: prothrombin fragment 1 binding was favoured by low calcium concentration, while osteopontin bound at higher levels. Scanning electron microscopy of COM and COD digested with proteinase K and cathepsin D revealed superficial and internal texture, as wells as surface erosion, in crystals from centrifuged and filtered urine, thus confirming the presence of intracrystalline proteins. Such features were absent from crystals precipitated from ultrafiltered urine. CONCLUSION: Binding of osteopontin and prothrombin fragment 1 to calcium oxalate is dictated primarily by ambient calcium concentration. Each protein may inhibit urolithiasis by inhibiting crystallization of its preferred crystal habit, and by facilitating the intracellular disintegration and dissolution of crystals attached to and internalized by renal epithelial cells.     

Preoperative FLAC/Granulocyte-Colony-Stimulating Factor Chemotherapy for Stage II Breast Cancer: A Prospective Randomized Trial

Background: Preoperative chemotherapy for stage II breast cancer may reduce locoregional tumors and provides initial treatment for systemic micrometastases. We conducted a prospective, randomized trial to evaluate the ability of intensive preoperative chemotherapy to enhance the outcome of this approach.Methods: Patients with clinical stage II breast cancer (T2N0, T1N1, and T2N1) were prospectively randomized to receive either preoperative or postoperative chemotherapy with five 21-day cycles of fluorouracil, leucovorin calcium, doxorubicin, and cyclophosphamide (FLAC)/granulocyte-colony-stimulating factor. Local therapy consisted of modified radical mastectomy or segmentectomy/axillary dissection/breast radiotherapy, according to patient preference.Results: Fifty-three women were randomized (26 preoperative chemotherapy and 27 postoperative chemotherapy). The objective clinical response rate of the primary tumor to preoperative chemotherapy was 80%, and the pathologic complete response rate was 20%. Preoperative chemotherapy reduced the overall incidence and number of axillary lymph node metastases. There was no difference in the use of breast-conserving local therapy between the two treatment arms. There were 20 local/regional or distant recurrences (9 preoperative and 11 postoperative). There was no difference in the overall or disease-free survival between the preoperative and postoperative chemotherapy arms.Conclusions: Preoperative FLAC/granulocyte-colony-stimulating factor chemotherapy was effective against local/regional tumors in stage II breast cancer but was otherwise comparable to postoperative chemotherapy.     

Intracrystalline proteins and urolithiasis: a synchrotron X-ray diffraction study of calcium oxalate monohydrate.

The existence of intracrystalline proteins and amino acids in calcium oxalate monohydrate was demonstrated by X-ray synchrotron diffraction studies. Their presence has implications for the destruction of calcium oxalate crystals formed in the urinary tract and the prevention of kidney stones. INTRODUCTION: Although proteins are present in human kidney stones, their role in stone pathogenesis remains unknown. This investigation aimed to characterize the nature of the relationship between the organic and mineral phases in calcium oxalate monohydrate (COM) crystals grown in human urine and in aqueous solutions of proteins and amino acids to clarify the function of proteins in urolithiasis. METHODS: COM crystals were grown in human urine and in aqueous solutions containing either human prothrombin (PT), Tamm-Horsfall glycoprotein (THG), aspartic acid (Asp), aspartic acid dimer (AspAsp), glutamic acid (Glu), glutamic acid dimer (GluGlu), or gamma-carboxyglutamic acid (Gla). Controls consisted of COM crystals precipitated from pure inorganic solutions or from human urine that had been ultrafiltered to remove macromolecules. Synchrotron X-ray diffraction with Rietveld whole-pattern peak fitting and profile analysis was used to determine nonuniform crystal strain and crystallite size in polycrystalline samples. RESULTS: Crystals precipitated from ultrafiltered urine had lower nonuniform strain than those grown in urine or in aqueous PT solution. Nonuniform strain was much lower in crystals grown in distilled water or in the presence of THG. For the amino acids, the highest nonuniform strain was exhibited by crystals grown in Gla solution, followed by Glu. Crystallite size was inversely related to nonuniform strain, with the effect being significantly less for amino acids than for macromolecules. CONCLUSIONS: Selected proteins and amino acids associated with COM crystals are intracrystalline. Although their incorporation into the mineral bulk would be expected to affect the rate of crystal growth, they also have the potential to influence the phagocytosis and intracellular destruction of any crystals nucleated and trapped within the renal collecting system. Crystals impregnated with protein would be more susceptible to digestion by cellular proteases, which would provide access to the crystal core, thereby facilitating further proteolytic degradation and mineral dissolution. We therefore propose that intracrystalline proteins may constitute a natural form of defense against renal stone formation.     

Bridge to transplantation with a left ventricular assist device for systemic ventricular failure after Mustard procedure

In recent years the conservative techniques to treat degenerative mitral valve insufficiency have developed to such an extent mainly due to a better understanding of the physiology and pathology of the mitral valve and to the possibility to get predictable and satisfactory results. Still a challenge persists for the cardiac surgeon when he has to deal with complex reconstructions. The technique described seems to offer an even better surgical option for patients with complex lesions involving the posterior mitral leaflet, especially as far as the hemodynamic performance is concerned.     

Differential modulation of CD4 and CD8 T-cell proliferation by induction of nitric oxide synthesis in antigen presenting cells

BACKGROUND: On antigenic stimulation, CD4 T cells generally proliferate more readily than CD8 T cells. The purpose of the present experiments was to determine whether nitric oxide (NO) might differentially modulate CD4 vs. CD8 T-cell proliferation. METHODS: Various concentrations of C57BL/6 iNOS +/+ and -/- bone marrow (BM)-derived antigen presenting cells (APC) (obtained by culture in granulocyte-macrophage colony-stimulating factor [GM-CSF] and interleukin [IL]-4) were cultured with purified BALB/c CD4 or CD8 T cells. RESULTS: Proliferation of CD4 T cells was similar in the presence of both NO synthase (iNOS) +/+ and -/- APC, whereas CD8 T cell proliferation was inhibited at the higher concentrations of iNOS +/+ dendritic cells (DC), coincident with increased levels of NO in the culture supernatant. Analysis of cytokine levels revealed that more interferon (IFN)-gamma, a potent inducer of NO synthesis in many cell types, was present in CD8 T cell than in CD4 T-cell-APC cultures. Addition of IFN-gamma to CD4 T-cell-APC cultures resulted in induction of NO synthesis and inhibition of proliferation at higher levels of NO than that required to inhibit CD8 T cell proliferation. However, CD4 T-cell proliferation was moderately inhibited in the presence of lipopolysaccharide (LPS)-stimulated CD11c DC, coincident with production of IFN-gamma and induction of NO synthesis. CONCLUSIONS: These findings indicate that CD8 T-cell proliferation can be inhibited by lesser amounts of APC-derived NO than is necessary to inhibit CD4 T cell proliferation. NO synthesis was not initiated in CD4 T cell-DC cultures unless costimulatory molecules were up-regulated and IFN-gamma was produced.     

An assessment of peripheral vascular disease in patients with diabetic foot ulcer

BackgroundPeripheral vascular disease (PVD) is a chronic limb ischaemia caused by atherosclerosis of the peripheral arteries. Diabetes mellitus is a risk factor for this disease. The most common symptom of PVD is muscle pain in the lower limbs on exercise. In diabetes, pain perception may be blunted by the presence of peripheral neuropathy. Therefore, a patient with diabetes and PVD is more likely to present with an ischaemic ulcer or gangrene than a patient without diabetes. The use of ankle-brachial-pressure index (ABI) in the clinic and bedside provide a measure of blood flow to the ankle. This could help early detection, initiate early therapy and may thus reduce the risk of critical limb ischaemia and limb loss.ObjectiveThe purpose of this study is to evaluate the occurrence of peripheral vascular disease using ankle-brachial index in diabetic patients with and without foot ulcers and the risk factors associated with diabetic foot ulcer (DFU).MethodThis prospective study involved all type 2 DM patients with foot ulcer (DFU population) and those without foot ulcers (non-DFU population) seen in our hospital. Their demographic, clinical and laboratory parameters were noted and documented. Measurement of ABI was done using a portable hand held Doppler and ankle pressures < 0.9 is suggestive of PVD.ResultsA total of 74 patients were recruited. Males were 42 (56.8%) and females were 32 (43.2%). The mean age of the patients was 62.89 ± 10.66 years and the duration of diabetes was 7.61 ± 7.57 years. Forty-six (62.2%) presented with foot ulcer while 28 (37.8%) were without foot ulcer. Patients with PVD represented by ABI < 0.9 was DFU 31(76.4%) while in non-DFU it was 10 (13.4%). Multivariant analysis of variables associated with DFU in those with ABI < 0.9 showed correlation with tobacco use r = .235, p = 0.044; duration of diabetes r = ?.427; p = 0.001; and systolic blood pressure r = ?.301; p = 0.009.DiscussionThe occurrence of PVD determined by the absence of >2 pulses by palpation alone and using ABI was 25.7% and 55.4% respectively. This suggests that assessment by palpation is subjective while the use of Doppler is quantitative and more reliable. DFU patients with PVD showed a significant correlation with tobacco use, duration of diabetes and systolic blood pressure but not with dyslipidaemia.ConclusionThis study shows that these patients had risk factors for PVD. The use of hand held Doppler will aid early diagnosis of critical limb at risk of loss and help to prevent and reduce the high rate of limb loss in our patients.     

The Effect of Residential Aged Care Size,Ownership Model,and Multichain Affiliation on Resident Comfort and Symptom Management at the End of Life

Context

In most resource-rich countries, a large and growing proportion of older adults with complex needs will die while in a residential aged care (RAC) facility.