Calciphylaxis – a topical overview

'Calciphylaxis', a calcification syndrome associated with ischaemic cutaneous necrosis, is acquired naturally in humans in disease states. It is a life and limb-threatening complication, usually observed in patients with renal disease and secondary hyperparathyroidism, but known to occur in the absence of renal or parathyroid disease. The reported mortality rate, which ranges from 60-80%, relates to wound infection, sepsis and organ failure. It is a small-vessel vasculopathy, which is estimated to occur in about 4% of haemodialysis patients. Clinically, violaceous, reticulate areas of cutaneous necrosis and eschar may be evident, particularly in the extremities. In addition to the clinical picture, a raised calcium phosphorous product, an elevated parathyroid hormone level, radiographic evidence of vessel and soft-tissue calcification and the finding of mural calcification affecting small arteries and arterioles on histopathology help to confirm the diagnosis of this entity which generally has a poor prognosis. A high index of suspicion and an active multidisciplinary management approach, with rigorous attention to wound care and prevention of sepsis, are vital in the management of these patients. In this overview, we discuss the pathophysiology, clinical features and associations, risk factors, diagnosis and management issues relating to calciphylaxis.     

Mutations in the retinal guanylate cyclase (RETGC-1) gene in dominant cone-rod dystrophy

The dominant cone-rod dystrophy gene CORD6 has previously been mapped to within an 8 cM interval on chromosome 17p12-p13. The retinal- specific guanylate cyclase gene (RETGC-1), which maps to within this genetic interval and previously was implicated in Leber's congenital amaurosis, was screened for mutations within this family and in a panel of small families and individuals with various cone and cone- rod dystrophy phenotypes. A missense mutation (E837D) was identified in affected members of the CORD6 family, as well as a second missense mutation (R838C) in three other families with dominant cone-rod dystrophy. RETGC-1 is only the fourth gene to be implicated in cone-rod dystrophy and this is the first report of dominant mutations in this gene.      

Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.

Mutations in the lipopolysaccharide (LPS) of Shigella spp. result in attenuation of the bacteria in both in vitro and in vivo models of virulence, although the precise block in pathogenesis is not known. We isolated defined mutations in two genes, galU and rfe, which directly affect synthesis of the LPS of S. flexneri 2a, in order to determine more precisely the step in virulence at which LPS mutants are blocked. The galU and rfe mutants invaded HeLa cells but failed to generate the membrane protrusions (fireworks) characteristic of intracellular motility displayed by wild-type shigellae. Furthermore, the galU mutant was unable to form plaques on a confluent monolayer of eucaryotic cells and the rfe mutant generated only tiny plaques. These observations indicated that the mutants were blocked in their ability to spread from cell to cell. Western immunoblot analysis of expression of IcsA, the protein essential for intracellular motility and intercellular spread, demonstrated that both mutants synthesized IcsA, although they secreted less of the protein to the extracellular medium than did the wild-type parent. More strikingly, the LPS mutants showed aberrant surface localization of IcsA. Unlike the unipolar localization of IcsA seen in the wild-type parent, the galU mutant expressed the protein in a circumferential fashion. The rfe mutant had an intermediate phenotype in that it displayed some localization of IcsA at one pole while also showing diffuse localization around the bacterium. Given the known structures of the LPS of wild-type S. flexneri 2a, the rfe mutant, and the galU mutant, we hypothesize that the core and O-antigen components of LPS are critical elements in the correct unipolar localization of IcsA. These observations indicate a more precise role for LPS in Shigella pathogenesis.     

Bacteriophage Mu d1(Apr lac) generates vir-lac operon fusions in Shigella flexneri 2a.

Previous studies have demonstrated that expression of virulence in Shigella spp. is controlled by growth temperature. To study the regulation of virulence (vir) genes, we set out to develop a rapid, easily-assayed phenotype with which to measure expression of virulence. This report described a procedure for isolating vir-lac operon fusions in S. flexneri 2a by using the specialized transducing bacteriophage Mu d1(Apr lac) of Casadaban and Cohen (M. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1976). Mu d1(Apr lac) lysogens were isolated and screened for loss of virulence and for temperature-dependent expression of the lactose genes on Mu d1(Apr lac). A recombinant plasmid carrying the Mu immunity gene was also introduced into lysogens of interest to stabilize the Mu d1(Apr lac) insertion and prevent possible thermal induction at 37 degrees C. The mutant which we isolated failed to penetrate tissue culture cells in the assay for virulence and produced almost 15-fold more beta-galactosidase when grown at 37 degrees C than when grown at 30 degrees C. The site of insertion of Mu d1(Apr lac) in this strain was shown to be in the 140-megadalton plasmid pSf2a140, which is known to be associated with virulence. P1L4-mediated transduction of the insertion into a virulent recipient demonstrated genetic linkage of Mu d1(Apr lac) with loss of virulence and temperature-dependent expression of beta-galactosidase. All of these features fulfill the phenotype expected for a Mu d1(Apr lac)-induced vir-lac operon fusion. This mutant provides us with a means of measuring expression of a gene function required for virulence by assaying for beta-galactosidase. The insertion will also serve as a starting point for mapping of genes on pSf2a140 which are necessary for expression of virulence.     

Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid.

In this study, we examined the relationship between the virulence of Shigella flexneri 2a and the ability of strains of S. flexneri 2a to absorb Congo red. Spontaneous nonpigmented (i.e., unable to bind Congo red [Pcr-]) derivatives of a virulent, pigmented (Pcr+) strain of S. flexneri 2a were isolated and assayed for virulence as determined by their ability to invade epithelial cells. All Pcr- mutants examined lost the ability to invade epithelial cells and were thus avirulent. Agarose gel electrophoresis of plasmid DNA from these avirulent, Pcr- mutants showed that the majority of these strains had lost a plasmid band corresponding to a virulence-associated plasmid, pSf2a140. In many of the mutants, concomitant loss of pigmentation, virulence, and pSf2a140 was accompanied by the appearance of a new plasmid, smaller than pSf2a140. We believe these new plasmids to be deletion derivatives of pSf2a140 and that loss of pigmentation and loss of virulence are associated with deletions in pSf2a140. We transduced Pcr- mutants to Pcr+ and isolated transductants which suppressed the Pcr- phenotype. None of the Pcr+ transductants regained the ability to invade epithelial cells. Several suppressors of the Pcr- phenotype were identified as mutations in cell wall biosynthesis. These results support our belief that although pigmentation is usually associated with virulence, genetic determinants unrelated to virulence can also affect the ability of the cell to bind Congo red. Therefore, the ability of S. flexneri 2a to bind Congo red does not necessarily imply the ability to invade epithelial cells. However, loss of ability to bind Congo red is accompanied by loss of virulence.     

Comparison between testosterone oenanthate-induced azoospermia and oligozoospermia in a male contraceptive study. IV. Suppression of endogenous testicular and adrenal androgens

Administration of supraphysiological doses of testosterone to normal men causes inhibition of spermatogenesis, but while most become azoospermic, 30-55% maintain a low rate of spermatogenesis. We have investigated whether there are differences in endogenous androgen production, of testicular and adrenal origin, which may be related to the degree of suppression of spermatogenesis. Thirty-three healthy Caucasian men were given weekly i.m. injections of 200 mg testosterone oenanthate (TE), 18 became azoospermic, while 15 remained oligozoospermic. Urinary excretion of epitestosterone, a specific testicular product, was reduced to <10% of pretreatment values, with no differences between the groups. Similar results were obtained for other markers of testicular steroidogenesis. Urinary and plasma adrenal androgens were also reduced during TE treatment: a statistically significant decrease in both (P < 0.001 and P < 0.05 respectively) was seen in the azoospermic but not oligozoospermic responders. These results suggest that testicular steroidogenesis is decreased to <10% by the administration of supraphysiological doses of exogenous testosterone. Differences in the degree of ongoing steroidogenesis in the testis do not appear to account for incomplete suppression of spermatogenesis, thus differences in androgen metabolism may underlie this heterogeneous response. A small but significant reduction in secretion of adrenal androgens was also detectable, the relevance of which is unclear.      

A locus for autosomal dominant anterior polar cataract on chromosome 17p

Inherited cataract is a clinically and genetically heterogeneous disease. Here we report the identification of a new locus for an autosomal dominant anterior polar cataract on the short arm of chromosome 17. To map this new locus we performed genetic linkage analysis with microsatellite markers in a four-generation pedigree. After exclusion of seven candidate loci for cataract, we obtained significant positive LOD scores for markers D17S849 (Z = 4.01 / theta = 0.05) and D17S796 (Z = 4.17 / theta = 0.05). Multipoint analysis gave a maximum LOD score of 5.2 (theta max = 0.06) between these two markers. From haplotype analysis, the cataract locus lies in the 13 cM interval between markers D17S849 and D17S796. This study provides the first genetic mapping of an autosomal dominant anterior polar cataract.      

Differential expression of FMR1, FXR1 and FXR2 proteins in human brain and testis

Lack of expression of the fragile X mental retardation protein (FMRP) results in mental retardation and macroorchidism, seen as the major pathological symptoms in fragile X patients. FMRP is a cytoplasmic RNA- binding protein which cosediments with the 60S ribosomal subunit. Recently, two proteins homologous to FMRP were discovered: FXR1 and FXR2. These novel proteins interact with FMRP and with each other and they are also associated with the 60S ribosomal subunit. Here, we studied the expression pattern of the three proteins in brain and testis by immunohistochemistry. In adult brain, FMR1, FXR1 and FXR2 proteins are coexpressed in the cytoplasm of specific differentiated neurons only. However, we observed a different expression pattern in fetal brain as well as in adult and fetal testis, suggesting independent functions for the three proteins in those tissues during embryonic development and adult life.