Cellular dysfunction in the diabetic fibroblast: impairment in migration,vascular endothelial growth factor production,and response to hypoxia

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.     

Oral spore vaccine based on live attenuated nontoxinogenic Bacillus anthracis expressing recombinant mutant protective antigen

An attenuated nontoxinogenic nonencapsulated Bacillus anthracis spore vaccine expressing high levels of recombinant mutant protective antigen (PA), which upon subcutaneous immunization provided protection against a lethal B. anthracis challenge, was found to have the potential to serve also as an oral vaccine. Guinea pigs immunized per os with the recombinant spore vaccine were primed to B. anthracis vegetative antigens as well as to PA, yet only a fraction of the animals (30% to 50%) mounted a humoral response to all of these antigens. Protective immunity provided by per os immunization correlated with a threshold level of PA neutralizing antibody titers and was long-lasting. Protection conferred by per os immunization was attained when the vaccine was administered in the sporogenic form, which, unlike the vegetative cells, survived passage through the gastrointestinal tract. A comparison of immunization of unirradiated spores with immunization of gamma-irradiated spores demonstrated that germination and de novo synthesis of PA were prerequisites for mounting an immune protective response. Oral immunization of guinea pigs with attenuated B. anthracis spores resulted in a characteristic anti-PA immunoglobulin isotype profile (immunoglobulin [G1 IgG1] versus IgG2), as well as induction of specific anti-PA secretory IgA, indicating development of mucosal immunity.     

Marfan syndrome with aortic dissection and triple-barrel aorta.

A case of Marfan syndrome with spontaneously and subsequently developed dissections of the aorta, one in the form of triple-barrel aorta, three times corrected by grafts is described. The autopsy revealed "healed" and acute dissections in almost the entire aorta outside the grafts. "Healed" thoracoabdominal dissection had true lumen (with entry and re-entry intimal tears), and old false lumen and in addition in its distal portion of a triple-barrel aorta was formed (dissection of healed aortic dissection). Lethal adventitial rupture occurred in the portion with an old false lumen. Dissection of the left subclavian artery, the right common carotid artery, resulting in saccular aneurysm, and avulsion of the right renal artery were also found.     

Structure and diversity of class I antigen presenting molecules in the mouse

Sequence comparisons among class I genes provide insight into the nature and origins of diversity in the human and mouse MHC. The profiles of diversity among alleles and between different loci indicate that genetic interactions among class I genes generate sequence diversity in both species. Humans and mice differ in the extent that sequence transfer occurs between loci. In mice, sequences encoding the antigen binding domain show little evidence of locus specificity. A series of mouse class I mutants have been analyzed, providing strong evidence that interlocus gene conversion plays a significant role in the exchange of sequences among class I genes. A similar process is suspected in human class I and both mouse and human class II genes. However, the transfer of sequence among genes in these groups appears to occur predominantly between alleles and only to a minor extent between loci.     

Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent.     

Isolation ofPasteurella multocida in a patient with spontaneous peritonitis and liver cirrhosis

A case of spontaneous bacterial peritonitis caused by Pasteurella multocida in a 12 year old boy with previously undiscovered cirrhosis of the liver is reported. This case is discussed and related to eight published cases of spontaneous peritonitis caused by Pasteurella multocida in adults, seven with cirrhosis of the liver and/or alcohol abuse, and one with systemic lupus erythematosus complicated by membranoproliferative glomerulonephritis. It would appear that spontaneous bacterial peritonitis caused by Pasteurella multocida is not confined to adults with a history of alcohol abuse or cirrhosis of the liver, but can also affect children with non-alcoholic cirrhosis of the liver.     

Passive Heymann nephritis in pre- and post-natal rats

Passive Heymann nephritis (PHN) was induced in pre- and post-natal rats by a single intra-peritoneal injection of 0.2 ml of a rabbit anti-rat kidney fraction 3 (rKF3) antibody. Immune complex formation occurred only in those glomeruli or parts of glomeruli which were open to the circulation. Double staining of kidney sections for the glomerular nephritogenic antigen and rabbit IgG, 2 days after the injection of the antibody showed an identical distribution of both components in the glomeruli. In rats killed more than 4 days after the injection of the anti-rKF3 antibody, the nephritogenic antigen could be demonstrated in the subcapsular glomeruli, in the absence of rabbit IgG; and the same applied when the kidneys had reached maturity. When the injected antibody was expected to be present in the circulation, no nephritogenic antigen was demonstrated in the glomeruli in the absence of the heterologous IgG. These observations indicate that the nephritogenic antigen appears in the glomerulus at the same time as the glomerular capillary loops open to the circulation. Unlike PHN in the adult rat, the immune complexes in the glomeruli of neonatal rats do not persist longer than 84 days.     

Immunohistochemical study of nasopharyngeal carcinoma using monoclonal keratin antibodies

Nasopharyngeal carcinoma (NPC) provides a unique opportunity to evaluate distinctive epidemiologic features and a possible etiologic relationship with Epstein-Barr virus (EBV) in human malignancy. The lack of a uniformly accepted pathologic classification for NPC has limited the application of this data, although the World Health Organization (WHO) developed a classification that may solve this problem. Monoclonal keratin antibodies were used for staining of NPC for evaluation of its assistance in diagnosis and classification. In the present immunohistochemical study, monoclonal keratin antibodies, designated AE1, AE2, and AE3, and a polyclonal keratin antibody (RAK) were used for study of the presence of keratin in 121 cases of NPC obtained from China and the United States. AE1 monoclonal antibody, which recognizes keratin protein classes 56.5K, 50K, and 40K, was shown to be the most sensitive and specific for NPC tumor cells among the keratin antibodies studied. In addition, some different keratin expression patterns could be identified between different kinds of epithelium and different tumor groups, with possible relevance to the histogenesis of the histologic subtypes of NPC.     

Synaptogenesis and myelinogenesis in dissociated cerebral cells from rat embryo on polylysine coated surfaces

Summary The appearance of myelinated axons and establishment of synaptic contacts in dissociated cells from rat embryo cerebral hemispheres grown on polylysine precoated surfaces were studied by light and electron microscopy. Typical myelin lamellae ensheathing axons were observed as early as the eighth day. Immature synaptic profiles exhibiting slight membrane thickening and few synaptic vesicles were observed after seven days. At 3 weeks, mature synapses containing many synaptic vesicles and the typical irregular membrane thickening could be seen. It is suggested that the presence of polylysine may provide a support which enhances the attachment and subsequent establishment of contacts between neuronal and glial cells, resulting in the early onset of myelin formation.