Retrospective study of a TTR FAP cohort to modify NIS+7 for therapeutic trials.

Protein stabilization and oligonucleotide therapies are being tested in transthyretin amyloid polyneuropathy (TTR FAP) trials. From retrospective analysis of 97 untreated TTR FAP patients, we test the adequacy of Neuropathy Impairment Score+7 tests (NIS+7) and modifications to comprehensively score impairments for use in such therapeutic trials. Our data confirms that TTR FAP usually is a sensorimotor polyneuropathy with autonomic features which usually is symmetric, length dependent, lower limb predominant and progressive. NIS+7 adequately assesses weakness and muscle stretch reflexes without ceiling effects but not sensation loss, autonomic dysfunction or nerve conduction abnormalities. Three modifications of NIS+7 are suggested: 1) use of Smart Somatotopic Quantitative Sensation Testing (S ST QSTing); 2) choice of new autonomic assessments, e.g., sudomotor testing of distributed anatomical sites; and 3) use of only compound muscle action potential amplitudes (of ulnar, peroneal and tibial nerves) and sensory nerve action potentials of ulnar and sural nerve - than the previously recommended attributes suggested for the sensitive detection of diabetic sensorimotor polyneuropathy. These modifications of NIS+7 if used in therapeutic trials should improve characterization and quantification of sensation and autonomic impairment in TTR FAP and provide better nerve conduction tests.

Macrophage activation in type 1 diabetic patients with catheter obstruction during peritoneal insulin delivery with an implantable pump.

OBJECTIVE: The purpose of this study was to evaluate the activation of macrophages in type 1 diabetic patients during peritoneal insulin delivery with an implantable pump against two types of insulin: that which was collected from the pump reservoir and that which came straight fromn the bottle (i.e., vial insltlin). Macrophage activation was studied in patients with and without cathcter obstruction and compared with activation in healthy subjects. RESEARCH DESIGN AND METHODS: Human insulin (21 PH, 400 U/ml; Hoescht) was collected from the pump reservoir (Minimed) of diabetic patients with (n = 3) or without (n = 7) catheter obstruction, as assessed by histological examination of the catheter tip. Monocytes were obtained from venous blood samples from both kinds of diabetic patients and from healthy subjects (n = 5) and were differentiated into monocyte-derived macrophages in culture. Their chemotaxis and tumor necrosis factor-alpha (TNF-alpha) release were studied with respect to both types of insulin, as previously stated. Formyl-methionyl-leucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) were used as controls. RESULTS: Neither insulin recovered from the pump reservoir nor vial insulin proved chemotactic to macrophages from either healthy subjects or those diabetic patients with and without catheter obstruction. The migration toward fMLP of macrophages from patients presenting a catheter obstruction was significantly higher than that observed with macrophages from either diabetic patients without obstruction or healthy subjects, the chemotactic index (mean +/- SD) was 3.81 +/- 0.36 vs. 2.30 +/- 0.89 and 2.60 +/- 0.80, respectively (P < 0.05). LPS significantly stimulated the TNF-alpha secretion of macrophages from diabetic subjects with a catheter obstruction, whereas both native and reservoir-recovered insulin had no effect on this release (144.83 +/- 67.25 vs. 5.15 +/- 2.93 and 5.27 +/- 2.43 pg/ml, P < 0.001). CONCLUSIONS: The human insulin used in implantable pumps, regardless of how long it had remained in the pump reservoir, did not induce macrophage activation in diabetic patients treated through intraperitoneal insulin delivery. In some of these diabetic patients, catheter obstruction could be explained by their high capacity of macrophage chemotaxis.

Chemotaxis activation of peritoneal murine macrophages induced by the transplantation of free and encapsulated pancreatic rat islets.

The present study concerns the influence of the transplantation of free and encapsulated (AN69 membrane, Hospal) islets on the chemotaxis of peritoneal macrophages. Fifty free or encapsulated rat islets, cultured for 24 h, were transplanted in the peritoneal cavity of mice (n = 12). Three days after transplantation, the chemotaxis of peritoneal murine macrophages was tested towards formyl-methionyl-leucyl-phenylalanine (fMLP) and a culture medium conditioned for 3 days by free rat islets isolated from the same rat donor. In response to fMLP, the chemotactic indexes of macrophages from mice transplanted with free or encapsulated islets were 8.09 +/- 2.10 and 9.45 +/- 2.76, respectively. These values were significantly higher than those obtained when macrophages from untreated mice were tested (2.42 +/- 0.23; p < 0.01). In response to culture medium conditioned by free islets, the transplanted encapsulated islets failed to enhance macrophage chemotaxis (2.41 +/- 0.53) compared to transplanted free islets (7.00 +/- 2.63; p < 0.01). Thus, encapsulation decreased the specific chemotactic activity of peritoneal macrophages induced by free islet transplantation, probably by prohibiting the diffusion of chemoattractants.

Chemotactic activity of culture supernatants of free and encapsulated pancreatic rat islets towards peritoneal macrophages.

Longterm efficiency of encapsulated pancreatic islet transplantation is limited by macrophagic reaction at the surface of biocompatible membrane. The aim of this work was to investigate the influence of soluble factors released by free and encapsulated islets on macrophage chemotaxis. The culture mediums conditioned for 6 days by free and encapsulated rat islets were incubated with peritoneal murine, rat allo and syngenic macrophages to study their migration. Culture supernatants of rat fibroblasts and acinar cells, glucose-stimulated free rat islets and supernatants of free rat islets treated by heat and proteinase K were also tested for their chemotactic activity. Islets encapsulation decreased the chemotactic activity of culture medium conditioned for 6 days by free rat islets on murine (1.66 +/- 0.20 vs. 3.10 +/- 0.23; p < 0.001, n = 5) and rat allogenic macrophages (1.63 +/- 0.21 vs. 4.70 +/- 0.36; p < 0.001, n = 9). There was no migration of rat macrophages towards syngenic islets. Fibroblasts exhibited a very strong chemotactic effect as compared to acinar cells. Insulin was not involved in macrophage migration. Proteinase K treatment of culture supernatant of free rat islets totally inhibited the chemotactic activity. After heating at 56 degrees C and 100 degrees C, this activity was reduced to 41 +/- 7% and 32 +/- 5% of the initial activity, respectively. In conclusion, pancreatic islet stimulated macrophage migration by release of immunological specific proteins partly retained by macroencapsulation.

The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms.

All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli beta-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.

Light and electron microscopy study of carbohydrate antigens found in the electron-lucent layer of Pneumocystis carinii cysts.

The localization and biochemical nature of antigens found in the electron-lucent layer (ELL) of Pneumocystis carinii cysts using polyclonal rabbit antibodies are described. These antigens, specific for the cystic stages of the parasite, were shared by organisms from different hosts, suggesting that they represent functionally important components of the cyst cell wall. The binding sites were situated on an interwoven net of fibrils in the ELL produced by mild to strong proteolysis. Degradation of this residue by glucanase and chitinase confirms that this layer contains branched glucan and chitin. In contrast, the prompt susceptibility of the polysaccharide-rich ELL to proteolysis reveals that proteins are also relevant in building up the cyst-wall glucan skeleton. It is therefore concluded that the formation of the Pneumocystis cyst wall shows differences to the typical fungal cell-wall architecture. The taxonomical debate regarding this unique protist is ongoing, and consideration of these immunological and morphological findings may be useful for the study of the biology and phylogeny of Pneumocystis.

Targeting the secretory pathway of Toxoplasma gondii.

Little is known about the extent of conservation in the organization of the secretory pathway in organisms as different as prokaryotes, eukaryotes, and humans. The protozoan parasite Toxoplasma gondii allows easy genetic manipulations, and numerous vectors for selection of transgenic parasites have been developed. One approach to study the molecular mechanism of protein sorting and trafficking is the expression of foreign proteins. Here we describe the design and application of a vector that targets proteins to the secretory pathway of T. gondii and yields high-level expression of Escherichia coli reporter proteins. The general strategies and potential problems in expressing foreign proteins in T. gondii are discussed.

Cytotoxicity of peritoneal murine macrophages against encapsulated pancreatic rat islets: in vivo and in vitro studies.

The aim of this work was to study in vivo and in vitro the involvement of macrophages and interleukin-1beta (IL-1beta) in the necrosis of encapsulated islets during xenograft and to evaluate the immunoprotective efficiency of the AN69 membrane. In vivo, 6 days after implantation, 65% of the membrane surface of the devices containing the islets was colonized with macrophages compared with only 5% of the surface of the empty control devices. The morphological aspect of implanted islets was altered and their insulin release decreased significantly compared with freshly isolated ones (265 +/- 50 vs. 507 +/- 81 microU/mL). In vitro, the insulin release of encapsulated islets cultured for 2 days decreased to 32 and 28%, respectively, in the presence of IL-1beta and macrophages. The addition of anti-IL-1beta antibody to the co-culture of macrophages and islets did not modify this loss of functional activity. Furthermore, IL-1beta passed through the AN69 membrane. In conclusion, macrophages are involved in damaging encapsulated pancreatic islets and are probably partly responsible for islet transplantation failure.

HIV-1 envelope glycoprotein gp120 down-regulates CD4 expression in primary human macrophages through induction of endogenous tumour necrosis factor-alpha.

Among immunological abnormalities present in human immunodeficiency virus type 1 (HIV-1)-infected individuals are dysregulation of cytokine production and CD4 down-regulation in both T-helper cells and monocytes/macrophages. The HIV-1 envelope glycoprotein 120 (gp120) has the ability to induce different cytokines in peripheral blood mononuclear cells and in monocytes/macrophages in vitro which in some instances have been reported to down-regulate macrophage CD4 expression. This study provides evidence that HIV-1 recombinant gp120 (rgp120) down-regulates both surface and total CD4 expression in primary tissue culture-differentiated macrophages (TCDM) at the level of transcription. The CD4 down-regulation observed in TCDM occurred between 6 and 12 hr after rgp120 treatment preceded by a peak of endogenous tumour necrosis factor-alpha (TNF-alpha) observed at 3-6 hr post-treatment. We demonstrate that the TCDM CD4 down-regulation observed after rgp120 treatment was inhibited by the use of an anti-huTNF-alpha monoclonal antibody (mAb), but not by mAb directed against other cytokines induced by rgp120, such as interleukin-1 beta (IL-1 beta) and interferon-alpha (IFN-alpha). The present findings roughly parallel those observed both in the sera of patients and in the monocytes/macrophages isolated from HIV-positive individuals, suggesting that gp120 by stimulating endogenous TNF-alpha production could be a good candidate for the CD4 down-regulation observed in the monocytes/macrophages of HIV-1-infected individuals. In contrast to CD4 down-regulation in HIV-infected lymphocytes, which results from a direct effect of viral genes on CD4 expression, soluble factors such as cytokines induced during HIV infection might explain the monocyte/macrophage CD4 dysregulation observed in acquired immune deficiency syndrome.

Trypanosoma cruzi in wild raccoons and opossums in North Carolina.

Trypanosoma cruzi was isolated from 1 of 12 (8.3%) opossums and 3 of 20 (15%) raccoons from the piedmont area of North Carolina. Although T. cruzi has been isolated previously from wild mammals in the southern United States, the present study is the first published report of naturally occurring T. cruzi infection of wild mammals in North Carolina. All 4 isolates were maintained successfully in axenic culture and in murine fibroblasts. In addition, intraperitoneal injection of 1 x 10(6) culture forms of 1 of the opossum isolates into C3H mice resulted in low but detectable parasitemias as early as day 6 of infection. These mice resolved parasitemia and survived infection. Intraperitoneal injection of 1 x 10(6) culture forms of a raccoon isolate resulted in the death of 3 out of 4 mice. Surprisingly, parasitemias were never detected in the peripheral blood of these mice. Infection of murine fibroblasts in vitro resulted in the presence of intracellular amastigote stages characteristic of T. cruzi.