Interaction of concanavalin a with bacterial lipopolysaccharides in agarose gel.

Binding of fluorescein isothiocyanate-labeled concanavalin A to a series of molecular species of lipopolysaccharide (LPS), purified from pathogenic bacteria, was studied via agarose gel precipitation experiments and the results were compared with available structural data.The LPS species could be divided into ConA-reactive and non-reactive ones. Reactivity resided in the O-specific chain of LPS, and binding to the lipid A or core moieties of LPS could not be demonstrated by the present methods. The α-D-glucose or α-D-mannose residues of the repeating O-specific oligosaccharide units appeared to be recognized by ConA, except when blocked by steric hindrance. Specificity of the reaction was verified by inhibition with 2% D-glucose. Binding by bacterium-specific sugar-residues could not be demonstrated.For precipitation to occur, polyvalency was required both for LPS and ConA, and the resulting precipitation appeared to be promoted by hydrophobic interactions between the lipid A moieties of LPS molecules. The LPS species were differently retained by the agarose gel, which can be explained by differences in their micellar structure in aqueous solution. E. coli O83 LPS did not readily diffused in 1% agarose gel, but its precipitation with ConA could be demonstrated either at elevated temperature or mixing it previously with molten agarose (Mancini's arrangement).

Lupus nephritis reoccurs following transplantation in the lupus prone mouse.

The incidence and pathomechanism of recurrent lupus nephritis (RLN) after transplantation is not clearly understood. Burning out of the autoimmune process or local immunoregulatory mechanisms in the kidney may be responsible for the low incidence of recurrence. These mechanisms cannot be investigated in human subjects, due to post-transplant immunosuppression. To investigate the pathomechanisms of RLN, male and female kidneys were transplanted from FAS deficient lupus prone (LPR) or control (FAS intact) MRL mice into either LPR or MRL recipients. Urinary protein and blood urea were assessed. Double negative (DN) lymphocyte proliferation was determined by flow cytometry. Two months after transplantation inflammatory infiltration of the glomerular, vascular and interstitial compartments were determined. Renal function as demonstrated by blood urea levels was normal in MRL recipients, but elevated in LPR recipients, independent of the donor strain. Paralleling functional results, inflammatory infiltration was mild or absent in MRL recipients of MRL grafts, and mild to moderate in MRL recipients of LPR grafts, suggesting that kidney removal from the autoimmune (LPR) environment significantly reduced inflammation. Graft infiltration was most severe in LPR recipients: grafts were similarly inflamed independent of the donor. All LPR recipients had significantly less CD4+ Th cells versus MRL mice. Transplantation of LPR grafts into MRL recipients reduced CD4+ Th cell percentage, accompanied by a slight induction of lupus autoantibody production. Our results demonstrate that lupus nephritis is not kidney specific in the LPR model with recurrence after transplantation in the absence of immunosuppression.

The phosphodiesterase-5 inhibitor vardenafil improves cardiovascular dysfunction in experimental diabetes mellitus.

BACKGROUND AND PURPOSE: Patients with diabetes mellitus exhibit generalized endothelial and cardiac dysfunction with decreased nitric oxide production. Elevated intracellular cyclic guanosine monophosphate (cGMP) levels contribute to an effective cardioprotection in different pathophysiological conditions. In this study, we investigated whether chronic treatment with the phosphodiesterase-5 inhibitor vardenafil could improve diabetic cardiovascular dysfunction by up-regulating the nitric oxide-cGMP pathway in the vessel wall and myocardium. EXPERIMENTAL APPROACH: Diabetes was induced in young rats by a single intraperitoneal injection of streptozotocin (60 mg x kg(-1)). In the treatment group, vardenafil (10 mg x kg(-1) x day(-1)) was given orally for 8 weeks. Diabetic control animals received vehicle for the same time. Left ventricular pressure-volume relations were measured by using a microtip Millar pressure-volume conductance catheter, and indexes of contractility, such as the slope of end-systolic pressure-volume relationship (E(max)) and preload recruitable stroke work (PRSW), were calculated. In organ bath experiments for isometric tension with rings of isolated aortae, endothelium-dependent and independent vasorelaxation was investigated by using acetylcholine and sodium nitroprusside. KEY RESULTS: When compared with the non-diabetic controls, diabetic rats showed increased myocardial and vascular transforming growth factor-beta1 expression, impaired left ventricular contractility (impairment of E(max) by 53%, PRSW by 40%; P < 0.05) and vascular dysfunction. Treatment with vardenafil resulted in higher cGMP levels, reduced transforming growth factor-beta1 expression, significantly improved cardiac function (improvement of E(max) by 95%, PRSW by 69%; P < 0.05) and greater vasorelaxation to acetylcholine and sodium nitroprusside in aortae from diabetic animals. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that impaired vascular cGMP signalling contributes to the development of diabetic vascular and cardiac dysfunction, which can be prevented by chronic phosphodiesterase-5 inhibition.

Skin disease is prevented but nephritis is accelerated by multiple pregnancies in autoimmune MRL/LPR mice.

The role of pregnancy in the progression of systemic lupus erythematosus (SLE) is still poorly understood. We analysed the effect of repeated pregnancies in MRL/lpr mice, a murine model of SLE. Seven-week old female mice were used: multiparous mice underwent three consecutive pregnancies (M); age-matched virgin mice served as controls (V). Animals were harvested at 20 weeks of age. Skin lesions were characterized by hair loss and scabs in the dorsum of the neck. Virgin skins showed thickened dermis, fibrosis and mononuclear cell infiltrates, which were practically absent in M. This was accompanied by higher IFN-gamma and lower IL-10 mRNA expression levels in V compared to M skin. Plasma IFN-gamma protein levels were also upregulated in V versus M. However, survival and kidney function were dramatically reduced and accompanied by hypertension after multiple pregnancies. Kidney histology also showed markedly increased renal lesions in M. In contrast to plasma and skin levels, both IL-10 and IFN-gamma mRNA were lower in the kidneys of V versus M mice. Concluding our findings, the pathomechanisms of lupus kidney and skin disease may be regulated differently at the organ level during pregnancy. Both IFN-gamma and IL-10 may be important regulatory cytokines at the local level.

Correlations among prostatic biopsy results, transrectal ultrasound findings and PSA levels in diagnosing prostate adenocarcinoma.

OBJECTIVE: The objective of this study was to evaluate transrectal ultrasound (TRUS) findings and prostate-specific antigen (PSA) levels in relation to prostatic biopsy results and to analyze their individual and combined performances in diagnosing prostate adenocarcinoma (PAC). METHODS: Men (n=143) with PSA levels above 4 ng/ml underwent TRUS and randomized ultrasound-guided prostatic biopsy through the peripheral zone, including additional hypoechoic nodules biopsies, if they were noted on TRUS. Data related to TRUS, biopsy, and PSA level results were then correlated. RESULTS: A significant correlation between TRUS images suspicious for PAC and a biopsy-confirmed diagnosis of PAC, or between the lack of such images and a negative biopsy result, was not found. However, a significant correlation was found between positive biopsy results and PSA levels greater or equal to 10 ng/ml. The sensitivity of transrectal ultrasound in making a diagnosis of PAC was 63%, whereas its specificity was 73%. CONCLUSION: We conclude that while the separate performances of these examinations were not effective in diagnosing PAC, the integrated use of these methods was more adequate for making the diagnosis.