Early barriers to neonatal porcine islet engraftment in a dual transplant model.

Porcine islet xenografts have the potential to provide an inexhaustible source of islets for ß cell replacement. Proof-of-concept has been established in nonhuman primates. However, significant barriers to xenoislet transplantation remain, including the poorly understood instant blood-mediated inflammatory reaction and a thorough understanding of early xeno-specific immune responses. A paucity of data exist comparing xeno-specific immune responses with alloislet (AI) responses in primates. We recently developed a dual islet transplant model, which enables direct histologic comparison of early engraftment immunobiology. In this study, we investigate early immune responses to neonatal porcine islet (NPI) xenografts compared with rhesus islet allografts at 1 hour, 24 hours, and 7 days. Within the first 24 hours after intraportal infusion, we identified greater apoptosis (caspase 3 activity and TUNEL [terminal deoxynucleotidyl transferase dUTP nick end labeling])-positive cells) of NPIs compared with AIs. Macrophage infiltration was significantly greater at 24 hours compared with 1 hour in both NPI (wild-type) and AIs. At 7 days, IgM and macrophages were highly specific for NPIs (α1,3-galactosyltransferase knockout) compared with AIs. These findings demonstrate an augmented macrophage and antibody response toward xenografts compared with allografts. These data may inform future immune or genetic manipulations required to improve xenoislet engraftment.

Dual islet transplantation modeling of the instant blood-mediated inflammatory reaction.

Islet xenotransplantation is a potential treatment for diabetes without the limitations of tissue availability. Although successful experimentally, early islet loss remains substantial and attributed to an instant blood-mediated inflammatory reaction (IBMIR). This syndrome of islet destruction has been incompletely defined and characterization in pig-to-primate models has been hampered by logistical and statistical limitations of large animal studies. To further investigate IBMIR, we developed a novel in vivo dual islet transplant model to precisely characterize IBMIR as proof-of-concept that this model can serve to properly control experiments comparing modified xenoislet preparations. WT and α1,3-galactosyltransferase knockout (GTKO) neonatal porcine islets were studied in nonimmunosuppressed rhesus macaques. Inert polyethylene microspheres served as a control for the effects of portal embolization. Digital analysis of immunohistochemistry targeting IBMIR mediators was performed at 1 and 24 h after intraportal islet infusion. Early findings observed in transplanted islets include complement and antibody deposition, and infiltration by neutrophils, macrophages and platelets. Insulin, complement, antibody, neutrophils, macrophages and platelets were similar between GTKO and WT islets, with increasing macrophage infiltration at 24 h in both phenotypes. This model provides an objective and internally controlled study of distinct islet preparations and documents the temporal histology of IBMIR.

Intestinal permeability in irritable bowel syndrome patients: effects of NSAIDs.

Intestinal permeability and the effect of NSAIDs on permeability were investigated in 14 irritable bowel syndrome (IBS) patients and 15 healthy subjects. In the study, 24-h urinary recoveries of orally administered polyethylene glycols (PEGs 400, 1500, and 4000) were not significantly different in healthy subjects and IBS patients before or after NSAID ingestion. Lactulose mannitol ratios in healthy subjects and IBS patients were not significantly different. Only time-dependent monitoring of PEG excretion showed that NSAIDs enhanced intestinal permeability for PEG 4000 in healthy subjects (P = 0.050) and for PEGs 400, 1500, and 4000 in IBS patients (P = 0.012, P = 0.041, and P = 0.012, respectively). These results show that intestinal permeability in IBS patients is not different from that in healthy subjects; NSAIDs compromise intestinal permeability in IBS patients to a greater extent than in healthy subjects, which suggests that IBS is associated with an altered response of the intestinal barrier to noxious agents.

Identification of a stimulator of steroid hormone synthesis isolated from testis.

Gonadal steroidogenesis is regulated by pituitary gonadotropins and a locally produced, unidentified factor. A 70-kilodalton (kD) protein complex secreted from rat Sertoli cells was isolated. The complex, composed of 28- and 38-kD proteins, stimulated steroidogenesis by Leydig cells and ovarian granulosa cells in a dose-dependent and adenosine 3',5'-monophosphate-independent manner. The follicle-stimulating hormone-induced 28-kD protein appeared to be responsible for the bioactivity, but the 38-kD protein was indispensable for maximal activity. The 28- and 38-kD proteins were shown to be identical to the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the proenzyme form of cathepsin L, respectively. Thus, a TIMP-1-procathepsin L complex is a potent activator of steroidogenesis and may regulate steroid concentrations and, thus, germ cell development in both males and females.

Diagnostic specificity of a real-time RT-PCR in cattle for foot-and-mouth disease and swine for foot-and-mouth disease and classical swine fever based on non-invasive specimen collection.

Foot-and-mouth disease virus (FMDV) and classical swine fever virus (CSFV) are highly contagious and can cause great economic losses when introduced into disease-free regions. Accurate estimates of diagnostic specificity (Sp) are important when considering the implementation of surveillance for these agents. The purpose of this study was to estimate diagnostic Sp of a real-time reverse-transcriptase PCR assay developed for detection of FMDV in cattle and domestic swine and CSFV in domestic swine based on non-invasive specimen collection. One thousand and eighty-eight range beef cattle were sampled from thirteen geographic locations throughout Texas. One thousand and one hundred market hogs and cull sows were sampled. Results for both FMDV and CSFV were considered positive if amplification occurred at or before 40 PCR cycles, inconclusive between 40 and 45 cycles and negative otherwise. Ten cattle had nonspecific PCR amplifications for FMDV, but none were classified as positive and only one as inconclusive. Specificity (95% confidence interval) was estimated as 100% (99.7, 100). There were 19 nonspecific PCR amplifications for FMDV in sampled swine with 1 classified as positive, 6 as inconclusive, and 12 as negative. Specificity (95% confidence interval) was estimated as 99.9% (99.5, 100). There were 21 nonspecific PCR amplifications for CSFV, and 1 was classified as positive. Specificity (95% confidence interval) was estimated as 99.9% (99.5, 100). These assays have high Sp, but nonspecific PCR amplifications can occur.

Repair of protease-damaged elastin in neonatal rat aortic smooth muscle cell cultures.

The objective of this study was to investigate the elastin repair process in the rat aortic smooth muscle cell culture after proteolytic injury. Although little studied in vivo, elastin repair is thought to occur through a sequential process involving enzymatic removal (debridement) of damaged fibers followed by synthesis of tropoelastin, its subsequent processing, and eventual incorporation into new insoluble elastin. A second repair mechanism of proteolytically damaged elastin in a culture system is reported here. Repair in this system relates directly to restoration of resistance to elastin solubilization by hot alkali. As expected, severe injuries were observed with porcine pancreatic elastase (PPE). Using PPE, only 6% of the elastin, relative to control, was resistant to hot alkali immediately after elastase treatment. 4 wk later, resistance to hot alkali had increased dramatically to a mean of 90%. Repair took longer after injury with 75 micrograms of PPE as compared with 50 micrograms of PPE. The limited elastic fiber proteolysis induced by either human neutrophil elastase or porcine trypsin was repaired in culture within 2 wk. Elastin that had been radiolabeled with [3H]lysine 4-5 wk before injury was converted from a hot NaOH-susceptible to a NaOH-resistant elastin fraction during recovery from PPE injury. At the same time, the frayed elastic fibers that were seen with the electron microscope immediately after PPE treatment were replaced by continuous bands of elastin that resembled those in control cultures. Restoration of NaOH resistance did not require a net increase in total cell layer elastin, suggesting that relatively little new tropoelastin incorporation into the cell layer was required for this type of repair. These results suggested a salvage repair mechanism for proteolytically damaged elastin.

Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene.

Angiotensin-converting enzyme (ACE) is a zinc-containing dipeptidyl carboxypeptidase that catalyzes the conversion of angiotensin I to the potent vasoconstrictor angiotensin II. By analyzing cDNA and genomic DNA, we have constructed a consensus sequence encoding the testis isozyme of mouse ACE. Testis ACE cDNA contains 2,435 base pairs and encodes a protein of 732 amino acids. The N-terminal 66 amino acids are unique to the testis isozyme, while the remaining 666 are identical to the carboxyl half of mouse somatic ACE. The overall conservation of amino acid sequence between the testis isozymes of the mouse, rabbit, and human is 78 to 84%. The conservation of amino acids for the N-terminal domain uniquely expressed within the testis is 63 to 67% between these species. Primer extension and RNase protection experiments show that RNA transcription of the testis ACE isozyme begins 16 or 17 bases upstream from the translation start site. A sequence element resembling a TATA box is found 25 bases 5' of the transcription start site. To create its unique isozyme of ACE, the testis begins mRNA transcription in the middle of the exonic-intronic structure of somatic ACE, within a sequence treated as an intron by somatic tissues. Testis ACE is not the result of alternative RNA splicing but seems due to the start of transcription at a unique site within the ACE gene.

Age of first exposure to American football and long-term neuropsychiatric and cognitive outcomes.

Previous research suggests that age of first exposure (AFE) to football before age 12 may have long-term clinical implications; however, this relationship has only been examined in small samples of former professional football players. We examined the association between AFE to football and behavior, mood and cognition in a large cohort of former amateur and professional football players. The sample included 214 former football players without other contact sport history. Participants completed the Brief Test of Adult Cognition by Telephone (BTACT), and self-reported measures of executive function and behavioral regulation (Behavior Rating Inventory of Executive Function-Adult Version Metacognition Index (MI), Behavioral Regulation Index (BRI)), depression (Center for Epidemiologic Studies Depression Scale (CES-D)) and apathy (Apathy Evaluation Scale (AES)). Outcomes were continuous and dichotomized as clinically impaired. AFE was dichotomized into <12 and ⩾12, and examined continuously. Multivariate mixed-effect regressions controlling for age, education and duration of play showed AFE to football before age 12 corresponded with >2 × increased odds for clinically impaired scores on all measures but BTACT: (odds ratio (OR), 95% confidence interval (CI): BRI, 2.16,1.19-3.91; MI, 2.10,1.17-3.76; CES-D, 3.08,1.65-5.76; AES, 2.39,1.32-4.32). Younger AFE predicted increased odds for clinical impairment on the AES (OR, 95% CI: 0.86, 0.76-0.97) and CES-D (OR, 95% CI: 0.85, 0.74-0.97). There was no interaction between AFE and highest level of play. Younger AFE to football, before age 12 in particular, was associated with increased odds for impairment in self-reported neuropsychiatric and executive function in 214 former American football players. Longitudinal studies will inform youth football policy and safety decisions.

A latent Mr 94,000 gelatin-degrading metalloprotease induced during differentiation of HL-60 promyelocytic leukemia cells: a member of the collagenase family of enzymes.

The treatment of HL-60 promyelocytic leukemia cells with phorbol esters (12-O-tetradecanoylphorbol-13-acetate) results in the appearance of cell substrate adhesion and the release of a Mr 94,000 gelatin-degrading metalloprotease. The appearance of the metalloprotease in the culture medium directly correlates with the timing and extent of cell substrate adhesion over a 24-h period. Anti-Mr 94,000 metalloprotease blocking antibodies were unable to interfere with the HL-60 cell substrate adhesion induced by 12-O-tetradecanoylphorbol-13-acetate, although they were able to specifically remove the Mr 94,000 gelatin-degrading activity from either HL-60 or U-937 cell-conditioned medium. A purified metalloprotease preparation was found to be predominantly latent and activated by organomercurials, acid treatment (pH 2 to 3.6), or 8 M urea. The activating effect of the latter two denaturing treatments suggests that conformational changes may be the common activating mechanism. The different treatments also caused the appearance of lower molecular weight gelatin-degrading bands (in gelatin zymogram gels) in a manner consistent with the autocatalytic cleavage that occurs with other collagnase proenzymes during activation. Edman degradation of a cyanogen bromide fragment from the Mr 94,000 metalloprotease provided the amino acid sequence [PR(C)GVPD] which is present in type I collagenase, stromelysin, and transin proenzyme sequences and partially conserved (V----N substitution) in the type IV collagenase proenzyme. This sequence has been reported to be important in the maintenance of the latent state of the transin proenzyme (R. Sanchez-Lopez et al., J. Biol. Chem., 263: 1892-11899, 1988) and is a sequence unique to collagenase proenzymes. The N-terminal sequence of the Mr 94,000 metalloprotease (AP-QDQST) is unique and distinct from other collagenases. Thus, the Mr 94,000 metalloprotease from HL-60 cells appears to be a distinct and new member of the collagenase family of proteases.

Anionic currents of chick sensory neurons are affected by a phospholipase A2 purified from the venom of the taipan snake.

A neurotoxic phospholipase A2 was purified from the venom of the taipan snake Oxyuranus scutellatus scutellatus by three consecutive chromatographic steps on ion exchange resins, followed by an affinity column prepared with a phosphatidylcholine derivative attached to Sepharose. The phospholipase was shown to be of type A2 (specific activity of 85 units/mg protein), and an apparent molecular weight of 16,000. Amino acid analysis shows the presence of approx. 150 residues with the N-terminal amino acid sequence: NLAQFGFMIRCANGGSRSALDYADYGC, different from all the phospholipases described until now. This enzyme is lethal to experimental mice (LD50 = 10 micrograms/20 g mouse weight) and affects ionic currents in chick (Gallus domesticus) dorsal root ganglion cells, measured by the whole-cell clamp technique. In symmetrical external/internal ionic solutions, after suppression of Na+, K+ and Ca2+ currents, external application of phospholipase at a low concentration (30 nM) was shown to increase the baseline current in a reversible manner. The augmented response was voltage-dependent and the effect was much greater for negative currents. In the presence of a salt gradient across the membrane (out 40 mM NaCl/in 140 mM CsCl), the current reversal potential revealed a shift in the positive direction typically due to Cl- ion flux through the membrane. External application of a 50 microM concentration of picrotoxin caused a reversible reduction of the phospholipase-induced chloride current. Moreover, no appreciable current block was detected after addition of 50 microM DIDS.

A novel high-density lipoprotein particle and associated protein in rat plasma.

This report describes the characterization of a novel rat apolipoprotein, which, as partial sequencing suggests, does not correspond to any described protein. The protein (termed PX) has an estimated molecular mass of 19.5 kDa and pI in the range 5.5-5.8. Monoclonal antibodies were obtained against protein PX and results on distribution among rat lipoproteins show it to be associated mainly with high-density lipoproteins (HDL), but also with VLDL. Immunoaffinity chromatography of total HDL shows protein PX to be included in a distinct lipoprotein particle, particularly enriched in free cholesterol, with which only traces of other apolipoproteins are associated. Immunologically crossreacting entities are found in the plasma of several species, including man. Retention of the epitope carried by the protein PX would suggest that it is of particular structural or functional importance. It remains to be established whether its function is associated with lipid metabolism.

Primary local orbital amyloidosis: biochemical identification of the immunoglobulin light chain kappaIII subtype in a small formalin fixed, paraffin wax embedded tissue sample.

BACKGROUND: Amyloidosis refers to a heterogeneous group of disorders associated with the deposition of chemically distinct amyloid fibril proteins. Precise determination of chemical amyloid type has diagnostic, therapeutic, and prognostic relevance. Although immunohistochemical techniques are used routinely to determine the amyloid type, the results can be negative or inconclusive, so that biochemical characterisation is often required. The development and application of new biochemical microtechniques suitable for examination of extremely small tissue samples is essential for precise identification of the deposited amyloid proteins. AIMS: To investigate biochemically the amyloid proteins present in a formalin fixed paraffin wax embedded orbital tissue from a patient with localised orbital amyloidosis in whom immunohistochemistry was not helpful in the determination of amyloid type. METHODS: Extraction of amyloid proteins from fixed tissue and their identification was carried out by a recently developed microtechnique. An extremely small tissue sample was dewaxed and extracted with formic acid. The extracted material was analysed using electrophoresis, western blotting, and amino acid sequencing. RESULTS: Biochemical examination of the extracted proteins showed the presence of immunoglobulin (Ig) derived amyloid proteins, which were composed of the N-terminal fragments of the Ig light chain kappaIII subtype (AL-kappaIII) (16, 8, and 3 kDa). CONCLUSIONS: This is the first chemically proved AL case reported in association with primary localised orbital amyloidosis. The biochemical microtechnique used was useful in achieving a precise diagnosis of amyloid disease, in a case where the results of routine immunohistochemical examination of amyloid were inconclusive.

Small bowel motility affects glucose absorption in a healthy man.

OBJECTIVE: To investigate the relationship between duodenojejunal motor activity and glucose absorption and to evaluate the effect of modification of duodenojejunal motility on glucose absorption by using the prokinetic drug cisapride. RESEARCH DESIGN AND METHODS: We examined seven healthy males, mean age 22 years, who were treated with cisapride 10 mg t.i.d. and placebo during 3 days in a randomized order, with a 2-week time interval. Duodenojejunal manometry was performed after each treatment on the morning of day 3, using an 18-lumen catheter. A liquid nutrient (3 kcal/min) was administered intraduodenally for 30 min, followed by a bolus of the glucose analog 3-O-methylglucose (3-OMG). Plasma 3-OMG concentrations were measured to assess absorption kinetics. RESULTS: The area under the 3-OMG concentration curve in the first 30 min after infusion was related to the number of antegrade propagated pressure waves (r = 0.49, P < 0.05), but not to the peak concentration, time to peak, and absorption fraction. The mean amplitude of pressure waves was higher during cisapride than placebo (P < 0.05), but the reoccurrence of interdigestive motility, numbers of pressure waves, and propagated pressure waves, as well as 3-OMG absorption characteristics, were not significantly different between the two treatments. During both treatments >60% of antegrade propagated pressure waves were propagated over a very short distance (1.5 cm). CONCLUSIONS: Glucose absorption in the human small intestine is related to short-traveling propagated intestinal contractile activity. Cisapride increases the amplitude of pressure waves, but does not affect the organization of pressure waves or the absorption of 3-OMG.

The identification of eight novel glucocerebrosidase (GBA) mutations in patients with Gaucher disease.

Mutations in the gene encoding for the lysosomal enzyme glucocerebrosidase (GBA) result in Gaucher disease. In this study, seven novel missense mutations in the glucocerebrosidase gene (A136E, H162P, K198E, Y205C, F251L, Q350X and I402F) and a splice site mutation (IVS10+2T-->A) were identified by direct sequencing of three amplified segments of the glucocerebrosidase gene. Five of the novel mutations were found in patients with neuronopathic forms of Gaucher disease, two of which, K198E and F251L, appear to be associated with type 2 Gaucher disease.

The characterization and sequence analysis of thirty CTG-repeat containing genomic cosmid clones.

We have systematically isolated and characterized DNA containing large CTG (n > 7) repeats from a human cosmid genomic DNA library. Using a CTG10 probe, more than 100 cosmid clones were identified, and 30 of these have been extensively characterized. The sequenced cosmids contain repeats that are between three and 19 perfect units (average 10 perfect repeats). The cosmids map to at least 12 different chromosomes. Sequence analysis of flanking regions suggests that more than one third of the repeats occur in exons, and many share strong sequence identity with databank sequences, including the gene involved in dentatorubral pallidoluysian atrophy (DRPLA). Genotyping of human DNA samples demonstrates that more than half of the repeats are polymorphic. This and similar collections of clones containing trinucleotide repeats should aid in the identification of genes that may contain expansions of trinucleotide repeats involved in human disease.

The structure and expression of the human neuroligin-3 gene.

The neuroligins are a family of proteins that are thought to mediate cell to cell interactions between neurons. During the sequencing at an Xq13 locus associated with a mental retardation syndrome in some studies, we discovered a portion of the human orthologue of the rat neuroligin-3 gene. We now report the structure and the expression of that gene. The gene spans approximately 30kb and contains eight exons. Unlike the rat gene, it codes for at least two mRNAs and at least one of which is expressed outside the CNS. Interestingly, the putative promoter for the gene overlaps the last exon of the neighboring HOPA gene and is located less than 1kb from an OPA element in which a polymorphism associated with mental retardation is found. These findings suggest a possible role for the neuroligin gene in mental retardation and that the role of the gene in humans may differ from its role in rats.

Primary structure determination and cloning of the cDNA encoding toxin 4 of the scorpion Centruroides noxius Hoffmann.

A peptide (toxin II-10), shown to be a Na+ channel blocker, was purified from the venom of the scorpion Centruroides noxius Hoffmann and sequenced by Edman degradation. It has 66 amino acid residues with the C-terminal residue (asparagine) amidated, as demonstrated by mass spectrometry. In addition, we report the cloning and the nucleotide sequence of the cDNA (CngtV) that codes for this toxin. We discuss the mechanism for processing the precursor peptide to its final form and compare the primary structure to that of other Na+ channel toxins. Two distinct groups of toxins seem to emerge from this comparison, suggesting a structure-function relationship of these peptides towards the recognition of either mammalian or insect tissues.

CD4 receptor binding peptides that block HIV infectivity cause human monocyte chemotaxis. Relationship to vasoactive intestinal polypeptide.

The octapeptide Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr (peptide T) and two structural analogs are potent agonists of human monocyte chemotaxis, evincing identical rank potency orders as was previously shown for their inhibition of human immunodeficiency virus (HIV) envelope binding and T cell infectivity. Chemotactic activity could be inhibited by anti-CD4 monoclonal antibodies (Mabs), but not other mononuclear cell Mabs. The core peptide required for chemotactic activity is a pentapeptide related to the sequence Thr-Thr-Asn-Tyr-Thr. Homologous pentapeptides, identified by computer search, were detected in several other non-HIV-related viruses as well as the neuropeptide vasoactive intestinal polypeptide (VIP). The CD4 molecule, therefore, appears to be a recognition molecule for a small signal peptide ligand whose active sequence is a homolog of peptide T and which may be the neuropeptide VIP.

Charybdotoxin and noxiustoxin, two homologous peptide inhibitors of the K+ (Ca2+) channel.

We show that noxiustoxin (NTX), like charybdotoxin (CTX) described by others, affects Ca2+-activated K+ channels of skeletal muscle (K+(Ca2+) channels). Chemical characterization of CTX shows that it is similar to NTX. Although the amino-terminal amino acid of CTX is not readily available, the molecule was partially sequenced after CNBr cleavage. A decapeptide corresponding to the C-terminal region of NTX shows 60% homology to that of CTX, maintaining the cysteine residues at the same positions. While CTX blocks the K+ (Ca2+) channels with a Kd of 1-3 nM, for NTX it is approx. 450 nM. Both peptides can interact simultaneously with the same channel. NTX and CTX promise to be good tools for channel isolation.

The genomic region encoding toxin gamma from the scorpion Tityus serrulatus contains an intron.

The gene encoding toxin gamma from the scorpion, Tityus serrulatus, was amplified by PCR from genomic DNA employing synthetic oligonucleotides designed from the reported cDNA sequence. The nucleotide sequence of this gene reveals the presence of an intron of 475 base pairs (bp) which interrupts the region that encodes the signal peptide of the precursor toxin. A comparison of the intron boundary sequences of the gamma toxin gene with ones from other arachnid genes is also presented.