Diagnosis of acute cholecystitis: sensitivity of sonography, cholescintigraphy, and combined sonography-cholescintigraphy.

BACKGROUND: Radiographic diagnosis of acute cholecystitis can be established using ultrasonography (US), cholecystoscintigraphy (HIDA), or both. Although both modalities have been effective in diagnosing acute cholecystitis (AC), physicians from the emergency department and admitting surgeons continue to request both tests in an attempt to increase the diagnostic accuracy of AC. This article reports the institutional experience of a large tertiary care health care facility, with respect to the sensitivity of US, HIDA, and combined US and HIDA. STUDY DESIGN: We conducted a retrospective review of 132 patients diagnosed with AC who underwent laparoscopic cholecystectomy during the same hospitalization. Patients were stratified into three groups: Group 1 (Gp1, n = 50) included patients who underwent US alone, group 2 (Gp2, n = 28) included patients who underwent HIDA scan alone, and group 3 (Gp3, n = 54) included patients who underwent both US and HIDA. RESULTS: The three groups did not differ with respect to age, liver chemistry, time to operation, and hospital length of stay. The sensitivity of US, HIDA, and combined US/HIDA as diagnostic modalities for acute cholecystitis was referenced to histopathologic confirmation. Sensitivity was 24 of 50 (48%), 24 of 28 (86%), and 49 of 54 (90%) for US, HIDA, and the combination of US/HIDA, respectively. CONCLUSIONS: HIDA scan is a more sensitive test than US in diagnosing patients with AC. Based on the results of this study, we recommend that HIDA scan should be used as the first diagnostic modality in patients with suspected acute cholecystitis; US should be used to confirm the presence of gallbladder stones rather than to diagnose AC.

Rhinocerebral mucormycosis treated with amphotericin B colloidal dispersion in three patients.

Rhinocerebral mucormycosis (zygomycosis) primarily affects diabetic or immunosuppressed patients and typically progresses rapidly, necessitating surgical excision and antifungal therapy with amphotericin B. Large doses of amphotericin B are required for cure, causing significant renal toxicity. Amphotericin B colloidal dispersion (ABCD; Amphocil, Sequus Pharmaceuticals, Menlo Park, CA) is a 1:1 complex of cholesteryl sulfate and amphotericin B, which results in significant reduction of toxicity, especially nephrotoxicity. We describe three patients with life-threatening rhinocerebral mucormycosis treated with ABCD. All patients had high serum creatinine levels due to prior treatment with amphotericin B; these levels reverted to normal during treatment with ABCD. Two patients with diabetes mellitus were cured after receiving a combination of surgery and ABCD therapy. The third patient, who had myelodysplastic syndrome, had an initial good response, with cure of the fungal infection; however, he eventually died of his primary illness. To the best of our knowledge, this is the first detailed clinical description of the treatment of mucormycosis with ABCD.

Low temperature reversibly inhibits transport from tubular endosomes to a perinuclear, acidic compartment in African trypanosomes.

We have used electron microscopy and flow cytofluorimetry to study endocytosis and intracellular transport of fluid phase bovine serum albumen gold complexes and membrane bound concanavalin A through endosomal compartments of bloodstream forms of Trypanosoma brucei rhodesiense. Both markers were rapidly endocytosed from the flagellar pocket. Within 20 minutes at 37 degrees C the markers reached a large, vesicular, perinuclear compartment that stained heavily with the CB1 monoclonal antibody. Neither marker left the flagellar pocket and entered cells at 4 degrees C. When cells were incubated at 12 degrees C, both markers entered the cell and were transported to collecting tubules, a tubular endosomal compartment that receives endocytosed material from coated endocytic vesicles. However, no material was transported from collecting tubules to the late, perinuclear compartment at 12 degrees C. The morphology of collecting tubule membranes was specifically altered at 12 degrees C; tubules became shorter and were arrayed near the flagellar pocket. The morphological alteration and the block in transport of endocytic markers to the perinuclear compartment seen at 12 degrees C were reversed 10 minutes after cells were returned to 37 degrees C. We also used flow cytofluorimetric measurements of pH dependent fluorescence quenching to measure the pH of the terminal endocytic compartment. Fluoresceinated lectins accumulated in a terminal compartment with a pH of 6.0-6.1, a value considerably higher than that of mammalian lysosomes. Fluorescence from fluoresceinated lectins in this terminal endocytic compartment was dequenched when bloodstream forms were incubated in the presence of chloroquine.

Processing and transport of a lysosomal membrane glycoprotein is developmentally regulated in African trypanosomes.

We have used pulse-chase immunoprecipitations methods to study early post-translational processing of CBI-gp, a lysosomal membrane glycoprotein expressed by African trypanosomes, Rap67, a polyclonal antibody to CBI-gp, immunoprecipitated a 100-kDa glycoprotein, gp100, from both bloodstream forms (BF) and procyclic forms (PF) of Trypanosoma brucei gambiense immediately after a 5-min pulse with radiomethionine. N-Glycanase digestion released a 67-kDa core protein, p67, from gp100 of both life cycle forms V8 protease digestion of p67 from BF and PF yielded 13 identical methionyl peptides, suggesting that gp100 from both life cycle forms have very similar or identical p67 core molecules. In BF, gp 100 carried both endoglycosidase H (EndoH)-resistant and EndoH-sensitive, N-linked oligosaccharides immediately after labeling. In PF, all the N-linked sugars on gp100 were EndoH sensitive. In BF, gp100 chased progressively into slower migrating 150-180-kDa components that obtained the CBI epitope, traveled to the cell surface where they could be biotinylated, and were proteolytically processed. The increase in mass of gp100 during chase in BF resulted from an elongation of N-linked oligosaccharides. Maturation of gp100 into 150-180-kDa CBI-gp was inhibited if BF were chased in the presence of glucosidase inhibitors castanospermine or deoxynojirimycin. In PF, gp100 did not increase in mass, could not be biotinylated on the cell surface, and was not proetolyzed during extended chases. Cryoimmunoelectron microscopy revealed that the antigens detected by rap67 are abundant in lysosomes and endosomes in both BF and PF. Thus, BF and PF express very similar or identical lysosomal membrane glycoproteins but process and transport them in very different ways.

Trypanosoma brucei brucei and T. b. gambiense: stumpy bloodstream forms express more CB1 epitope in endosomes and lysosomes than slender forms.

CB1-glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1-glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1-glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1-glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle.

Transport of a lysosomal membrane glycoprotein from the Golgi to endosomes and lysosomes via the cell surface in African trypanosomes.

gp57/42 is a membrane glycoprotein localized in the trans-Golgi, flagellar pocket region of the cell surface, endosomes and lysosomes of bloodstream forms of Trypanosoma brucei rhodesiense. Pulse-chase immunoprecipitation experiments revealed that gp57/42 acquires a unique N-linked oligosaccharide recognized by the CB1 monoclonal antibody 20-30 minutes after protein synthesis, probably in the trans-Golgi. We refer to gp57/42 molecules that carry the CB1 epitope as CB1-gp. Pulse labeled CB1-gp contained only one core protein, p57, when chase times were 30 minutes or less. As time of chase increased from 30 to 60 minutes, a new polypeptide, p42, appeared in N-glycanase-treated CB1 immunoprecipitates. Since p57 and p42 share 10 of 13 methionyl peptides, we conclude that p42 is a fragment of p57. Cleavage of p57 to p42 was not inhibited when cells were chased in two thiol protease inhibitors or in 3,4-diisocoumarin, but was inhibited by leupeptin. Cell surface biotinylation was used to determine if newly synthesized CB1-gp was transported from the Golgi to the surface. When cells were pulse labeled and chased for 30 minutes, as much as 40% of the radiolabeled CB1-gp could be biotinylated on the cell surface. The amount of CB1-gp that could be biotinylated decreased when chases were extended from 30 to 60 minutes, suggesting that pulse labeled CB1-gp left the surface. In contrast, pulse labeled variant surface glycoprotein molecules continued to accumulate on the surface where they could be biotinylated between 30 and 60 minutes of chase. Biotinylated CB1-gp derived from cells chased for 30 minutes contained p57 but no p42. However, when labeled cells were biotinylated after a 30 minute chase and then incubated another 30 minutes at 37 degrees C, the biotinylated CB1-gp contained both p57 and p42. The p57 in biotinylated CB1-gp was not cleaved to p42 if the additional incubation was done at 4 or 12 degrees C. This suggests that transport to a compartment where processing occurs and/or the processing enzymes are inhibited by low temperature. When surface biotinylation was done after a 60 minute chase, p42 was detected in biotinylated CB1-gp, suggesting that CB1-gp molecules had passed through the processing compartment and then appeared on the cell surface. Thus, a major portion of the newly synthesized CB1-gp is routed from the Golgi to endocytic compartments via the cell surface. In trypanosomes this process involves a unique surface domain, the flagellar pocket.(ABSTRACT TRUNCATED AT 400 WORDS)

Heparitinase inhibition of mesoderm induction and gastrulation in Xenopus laevis embryos.

We have examined the involvement of proteoglycan molecules in the induction of mesodermal tissue in Xenopus laevis embryos. Blastocoelic injections of the enzyme heparitinase at early blastula stages lead to gastrulation defects and to failures in the development of anterior embryonic structures. The period of sensitivity of embryos to this treatment suggests a possible role for these molecules during mesoderm induction. We show that heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans are the predominant sulfated glycoconjugates synthesized in early Xenopus embryos and that HSPGs are degraded by blastocoelic injections of heparitinase. Further, bFGF induction of mesoderm in explants of Xenopus stage 8 embryonic animal cap tissue is blocked by heparitinase but not by Chondroitinase ABC, using three separate criteria of mesoderm induction. Since HSPGs present in blastula animal cap cells are digested by heparitinase under the culture conditions used in the mesoderm-induction assay, we suggest that cell-surface heparan sulfate proteoglycans are required for basic fibroblast growth factor-mediated mesoderm induction.

Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote.

Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been identified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers. Trypanosomes are the most primitive eukaryote yet in which BiP has been identified. The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy. The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum. The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al. (1989) Biochemistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.

Trypanosoma brucei rhodesiense: membrane glycoproteins localized primarily in endosomes and lysosomes of bloodstream forms.

Bloodstream forms of Trypanosoma brucei rhodesiense take up macromolecules in endocytic vesicles that form in a large coated pit called the flagellar pocket. Glycoproteins that bind to ricin are concentrated in the flagellar pocket and in intracellular vesicles. We purified Triton X-100-soluble ricin-binding glycoproteins by lectin affinity chromatography and immunized mice to generate hybridomas. Monoclonal antibody produced by the CB1 hybridoma recognized heterodisperse trypanosome components migrating with M(r) 84-140 kDa in immunoblots. CB1 binding was specifically inhibited by lactose. The CB1-reactive material was purified by sequential affinity chromatography on ricin- and CB1-Sepharose. N-Glycosidase F, but not endoglycosidase H, digestion destroyed CB1-reactivity of purified material. This suggests that N-linked oligosaccharides contribute to the CB1 epitope. Glycosidase digestion of biosynthetically radiomethionine-labeled, affinity purified, CB1-reactive material yielded two radiolabeled polypeptides, p57 and p42. Thirteen methionyl peptides were resolved in one-dimensional peptide maps of V8 protease digests of p57; p42 had 10 methionyl peptides with mobilities indistinguishable from those of peptides of p57. This suggests that p57 and p42 are closely related. In cryoimmunoelectron microscopy studies CB1 specifically labeled the interior surface of tubular and vesicular membranes located between the nucleus and the flagellar pocket. These membranes were morphologically identical to structures that have been previously identified as trans Golgi, lysosomal, and endosomal elements. In double-labeling studies endocytosed serum albumen-gold complexes were found in the lumen of vesicles that had CB1-reactive material in their membranes. This provides direct evidence that vesicles containing high levels of CB1-reactive material are part of the lysosome/endosomal system. Some CB1-reactive material was also detected in the flagellar pocket by cryoimmunoelectron microscopy. Corrolated flow cytofluorimetry and immunofluorescence analysis showed that 85-96% of the total CB1-reactive material was intracellular and inaccessible to antibody in living cells. The 4-15% of the total CB1-reactive material accessible to antibody in living cells was localized in the flagellar pocket. Bloodstream forms of Trypanosoma brucei brucei, Trypanosoma brucei gambiense, and T.b. rhodesiense all expressed the CB1 epitope. However, expression of this epitope is developmentally regulated during the parasite life cycle, for no CB1-reactive material was detected in procyclic forms. The trypanosome proteins detected by CB1 show some similarities to vertebrate lysosomal and endosomal membrane proteins.

Trypanosoma brucei rhodesiense bloodstream forms: surface ricin-binding glycoproteins are localized exclusively in the flagellar pocket and the flagellar adhesion zone.

Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed.

Isotretinoin dermatitis simulating acute pityriasis rosea.

A pityriasis rosea-like eruption developed in two acne patients receiving isotretinoin and gradually resolved once the dosage was reduced. Histologically the lesions, which were characterized by psoriasiform hyperplasia, bore no resemblance to pityriasis rosea. We believe the eruptions were a side effect of isotretinoin therapy.