Biomedical subjects
Publications and source records attributed to W R Brown.
Tumoral calcinosis-like lesions of the foot are a pedal manifestation of end-stage renal disease. Although they are benign, they have the potential to cause significant morbidity because of their invasive nature. Following a brief description of tumoral calcinosis-like lesions, the authors provide an illustrative case presentation including radiographs, magnetic resonance images, surgical photographs, and histopathology.
We have been using alkaline phosphatase (AP) histochemical staining, formerly a research tool for the study of cerebral cortical vascular morphology, to examine pathological changes in the cortex and deep cerebral structures. Deep structures stain similarly to the cortex. The AP stain is found in the afferent vessels (small arteries, arterioles, and capillaries), but not in venules and veins. The stain is also present in leaky vessels, such as those in the area postrema. The vascular supply to the cerebrum is not homogeneous. Supply to the deep white matter, for instance, derives from the leptomeningeal border zone, and then medullary arterioles must wind their way for up to 4 cm before arriving at their ultimate destination. Adding to the difficulties, tortuosities develop in some of these vessels with aging. According to some calculations, hypertensive levels of blood pressure would be required to maintain irrigation through some of these vessels. We have identified a venous alteration that attends aging: periventricular venous collagenosis (PVC) is a previously unrecognized, noninflammatory, mural disease of the periventricular veins. In severe cases, examples can be found of veins that are completely occluded by this process. PVC is found in 65% of subjects over 60 years old, and it strongly correlates with leukoaraiosis. In addition to previously mentioned aging-related changes, we have found extreme tortuosity, multiplications, and aneurysms of the smallest arterioles and lumpy-bumpy capillaries in the deep structures of patients with Alzheimer's disease.
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Cloning a cDNA for human IgGFc binding protein (FcgammaBP) from human colonic epithelial cells reveals an mRNA and coding region of 17 and 16.2 kilobases, respectively. The predicted amino acid sequence contains 12 occurrences of a 400-amino acid cysteine-rich unit resembling that found in mucin. A motif (CGLCGN) in FcgammaBP is conserved in MUC2 and prepro-von Willebrand factor. The N-terminal 450-amino acid sequences are necessary and sufficient to confer IgG Fc binding activity. FcgammaBP mRNA is expressed only in placenta and colonic epithelial cells. These results suggest that FcgammaBP may play an important role in immune protection and inflammation in the intestines of primates.
We cloned and analyzed the gene (IGHG3) encoding cattle IgG3 using a spleen cDNA library and a genomic phage clone. IGHG3 differs most remarkably from IGHG1 and IGHG2 in the structure of its hinge. The IGHG3 hinge is encoded by two exons, one of which encodes a 22 amino acid 3' extension of the CH1 exon. The IGHG3 gene is highly homologous to the C-gamma pseudogene previously reported. Seventy-one DNA samples from different cattle were tested by pulsed-field gel electrophoresis and Southern blot to detect genetic polymorphisms. This analysis revealed two different alleles which we designated IGHG3(a) and IGHG3(b). The two putative alleles differ from each other by a 6 amino acid substitution in the coding region and an 84 base pair insertion in the intron between the CH2 and CH3 exons. Our data also indicate that the cattle heavy chain locus spans approximately 400 kilobases from IGHM to IGHA and encodes only three IgG subclasses, all of which are transcriptionally active.
The complete genomic sequence of a bovine C alpha gene is reported here. The genomic sequence was obtained from a C alpha phage clone that had been cloned from a genomic EMBL4 phage vector library. The C alpha sequence had previously been expressed as a chimeric antibody and identified as IgA using IgA-specific antibodies. Intron/exon boundaries were determined by comparison of the genomic sequence with an expressed bovine C alpha sequence obtained from spleen by reverse transcription-polymerase chain reaction (RT-PCR). Analysis of 50 Swedish bovine genomic DNA samples using genomic blots and five different restriction enzymes failed to detect evidence of polymorphism. However, PstI digests of Brown Swiss DNA showed a restriction fragment length polymorphism (RFLP), suggesting that at least two allelic variants of bovine IgA exist. Comparison of the deduced amino acid sequence of bovine IgA with sequences available for other species indicated that the highest homology was with that of swine, another artiodactyl. This was the highest homology observed for all mammalian IgA compared except for that between IgA1 and IgA2 in humans. Bovine IgA shares with rabbit IgA3 and IgA4, an additional N-linked glycosylation site at position 282. However, the collective data indicate that cattle are like swine and rodents and unlike rabbits in having a single locus of the gene encoding IgA of this species.
We have used gene targeting in the DT40 cell line to create a cell line which expresses a fusion between CENP-C and a mouse steroid receptor and which behaves as a conditional loss of function mutant of CENP-C. Under restrictive conditions these cells arrest at the metaphase/anaphase junction and after a delay of approximately 2.5 h die by apoptosis. These results indicate that CENP-C is either necessary for anaphase chromosome movement or for mediating a signal which triggers centromere function during anaphase. Our approach is simple and applicable to a wide range of proteins with general cell autonomous functions in vertebrates.
PURPOSE: To examine the vascular supply and architecture of the germinal matrix in the preterm neonatal brain and to determine whether veins or arterioles are the source of germinal matrix hemorrhage. METHODS: Brains from eight preterm neonates (24 to 35 weeks' gestation) and two full-term infants were fixed in alcohol, embedded in celloidin, sectioned at 100- and 500-micron thicknesses, stained for alkaline phosphatase, and examined with light microscopy. High-resolution contact radiographs of 500-micron-thick sections were also mounted on glass slides for microscopic examination. RESULTS: The upper and middle regions of the germinal matrix are supplied by branches of the lateral striate arteries, whereas the inferior part is supplied by branches of the recurrent artery of Heubner. In brain sections from four of the preterm infants, we found 15 circumscribed hemorrhagic foci within the germinal matrix. The largest was 5 mm in diameter; the smallest, 1 mm. All hemorrhages but one were closely associated with veins, with significant involvement of the perivenous space. The other hemorrhage appeared to be associated with an arteriole. In term and preterm infants, we found no arteriolar-to-arteriolar shunts, precapillary arteriolar-to-venules shunts, or vascular rete. At all gestational ages, the terminal vascular bed had only conventional branchings and connections. CONCLUSION: In preterm neonates, staining for endogenous alkaline phosphatase allows visual differentiation between afferent and efferent vessels. Germinal matrix hemorrhage in preterm neonates is primarily venous in origin. A hemorrhage can tunnel along the venous perivascular space, collapsing the vein and rupturing the tethered connecting tributaries. Extravasation of blood from the arterial circulation appears to be much less common.
We have used telomeric DNA to break two acrocentric derivatives of the human Y chromosome into mini-chromosomes that are small enough to be size- fractionated by pulsed-field gel electrophoresis. One of the mini-chromosomes is about 7 Mb in size and sequence-tagged site analysis of this molecule suggests that it corresponds to a simple truncation of the short arm of the Y chromosome. Five of the mini-chromosomes are derived from the long arm, are all rearranged by more than a simple truncation, and range in size from 4.0 Mb to 9 Mb. We have studied the mitotic stabilities of these mini-chromosomes and shown that they are stably maintained by cells proliferating in culture for about 100 cell divisions.
Monoclonal populations of mucosal T cells were established from the earliest visible lesions in eight patients with well defined Crohn's disease. The FACS phenotype of all the mucosal derived clones to date are TCR alpha/beta+, CD3+, CD4+, and CD45RO+ memory cells. TCR variable region Beta chain analysis revealed predominantly V beta families 1, 2, 5.1, 5.2, 6, 7 and 8, with V beta family analysis supporting antigen expansion in the diseased mucosa. Putative autoreactivity was evaluated by stimulating individual clones with a battery of antigens and determining proliferation and IL-2 production by thymidine incorporation at 72 h. Antigens tested included crude Crohn's diseased (CD) colon and small bowel homogenates, CD brush border preparations, crude CD colon and small bowel mucin, and purified CD small bowel mucin. Controls included clone, APC, tetanus toxoid and either PHA or Staphylococcus enterotoxin B. A total of 200 clones were studied with 29.5% or 59 clones demonstrating proliferation and/or IL-2 production. T cell receptor V beta gene usage evaluated in a small number of reactive clones correlated with the expanded patient families. Seven of the fifteen represented families revealed diverse T cell receptor gene use and no disease overlap.
Swine have the largest number of IgG subclass genes of all species so far studied but have a single gene for IgA which occurs in two allelic forms that differ in hinge length. Swine also have constant region genes for C mu and C epsilon, but lack a gene homologous to that which encodes IgD in rodents and primates, despite the otherwise high degree of sequence similarity of all other swine CH genes with those of humans. Swine have < 20 VH genes, a single JH and perhaps a limited number of DH segments. Newborn piglets show preferential VH and DH usage and may use gene conversion as a mechanism for expanding their antibody repertoire. Despite the close similarity of their Ig gene sequences to humans, swine belong to the group of animals that includes rabbits, chickens and cattle when classified on the basis of B cell development. This group, unlike rodents and humans, have a single VH family, use hindgut follicles early in life (rather than bone marrow throughout life) to diversify their antibody repertoire and probably all use gene conversion. It is proposed that IgD may serve a function in repertoire development in rodents and humans which is unnecessary in the chicken-lagomorph-artiodactyl group. The diversity of immunoglobulins and immunoglobulin genes among species justifies the quest of veterinary immunologists to define the system for their species of interest rather than making extrapolations from mouse and human immune systems.
A 34-year-old man with acquired immunodeficiency syndrome (AIDS) developed pneumatosis cystoides intestinalis associated with an intestinal infection with Cryptosporidium organisms. The pneumatosis resolved upon treatment of the cryptosporidiosis with paromomycin and clofazimine. Four other cases of benign pneumatosis cystoides intestinalis in adults with AIDS and associated cryptosporidiosis have been reported, which suggests that cryptosporidial infection may be pathogenetically involved in the pneumatosis and not merely incidental.
Following a 1994 collaborative research workshop on animal studies of the eosinophilia-myalgia syndrome (EMS), the Research Advisory Council (RAC) concluded that it would be instructive to have histopathologic slides from the different investigators and laboratories evaluated simultaneously using the same pathologist, pathologic criteria, and scoring system for cross-study comparisons of effects. The author evaluated the skin, underlying fascia and supporting tissues, and muscles, stained with hematoxylin and eosin, from 20 separate studies, representing 4 mouse strains, several protocols, and tissues from over 250 mice. The author concluded that, based on the material available, the findings are inconsistent, nonreproducible, and do not support a conclusion that L-tryptophan, case associated L-tryptophan, 1,1'-ethylidenebis(tryptophan), 3-phenylamino alanine, or a combination thereof, under the conditions of the studies, induce lesions of skin or muscle in the C57 Black, Balb/c, SJL, or B10.s strains of mice.
Recently published genomic and cDNA sequences for porcine IgA suggested that the splice acceptor site in the C alpha 1-C alpha 2 intron was an AA rather than an AG dinucleotide. This possibility was tested in an in vitro HeLa cell splicing system using an RNA substrate corresponding to the genomic DNA with the putative AA splice site. Data indicated that splicing occurred at a cryptic AG site 12 nucleotides into the C alpha 2 domain rather than at the AA site. The possibility that swine B cells could use either site was tested by preparing the cDNAs from 13 different samples representing nine animals and amplifying the segment from the first C alpha 1 nucleotide to nucleotide 532 in C alpha 2 (genomic DNA numbering system). Analysis on a 6% polyacrylamide sequencing gel revealed two polynucleotide products in most samples that differed by the expected 12 nucleotides, suggesting that swine could use both splice sites. Sequence analysis confirmed that the shorter form was spliced at the downstream site and the larger form at the apparent upstream AA site. However, when the genomic DNA from an animal expressing only the longer polynucleotide was cloned and sequenced, the upstream splice acceptor site was AG not AA. Thus the data suggested that porcine IgA occurred in two allelic forms, designated IgAa and IgAb, which differ by an apparent G to A mutation in the last nucleotide of intron 1 resulting in a short-hinged (two amino acids, IgAb) variant, in which the downstream cryptic splice site is used, as well as a "normal-hinged" (six amino acids, IgAa) variant. Evidence that IgAa and IgAb are allelic was confirmed by genotypic analyses of progeny from matings of IgAa/IgAb heterozygotes. Evidence that both transcripts are functional was confirmed by showing that serum IgA levels were similar in animals homozygous for each variant.
This study was undertaken to determine whether microemboli injected in a predetermined sequence would maintain that sequence once they came to rest in brain microvessels. If so, the injection of different-colored microspheres at different times could be used to bracket-in-time emboli that are known to be released into the circulation during cardiopulmonary bypass. We injected different-colored microspheres into the arterial circulation of anesthetized dogs before and after the injection of fat emboli and before and after cardiopulmonary bypass. Coronal slices of the dog brains were embedded in celloidin, sectioned at 100 microns, and stained for alkaline phosphatase. The afferent cerebrovasculature stained dark brown against a light background, and the proximal/distal orientation of many of the arterioles could be determined by following their course within the thick sections. When different types of emboli were found in a single arteriole, they appeared in the order injected or the order of occurrence in the bypass protocol in 99.3% of the 867 such arterioles counted. Therefore, the microemboli maintained their ordered sequence with only a very small degree of mixing. Once they came to rest, there was not sufficient collateral blood flow in the brain microvessels to move them into disordered positions. This dog model should facilitate studies of the time of release of microemboli within narrower windows of time during cardiopulmonary bypass.
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Emboli in brain tissue after cardiopulmonary bypass were reported in the literature 30 years ago, but there is little objective evidence confirming the presence of emboli in the brain after cardiopulmonary bypass with more modern equipment and techniques. Recently, with alkaline phosphatase vascular staining, we found an acellular fatty material in brain microvasculature from autopsy material of patients who died shortly after cardiopulmonary bypass. These fatty intravascular collections range in diameter from 10 to 70 microns, a size that lodges in the smallest vessels of the microvasculature. They have been found in numbers sufficient to cause detectable neurologic dysfunction and are believed, but not proved, to be emboli. By sequentially injecting colored microspheres, we can determine when emboli occur during experimental cardiopulmonary bypass. In ongoing related studies, magnetic resonance imaging was performed before cardiac valve replacement in 39 patients for whom preoperative and postoperative neurologic and neuropsychologic testing was available. Preliminary results suggest that magnetic resonance imaging evidence of prior stroke is not a significant risk factor for cognitive or motor decrement after cardiopulmonary bypass.
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