Chimeric Allografts Induced by Short-Term Treatment With Stem Cell-Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: A Study in Rats.

Transplant tolerance allowing the elimination of lifelong immunosuppression has been the goal of research for 60 years. The induction of mixed chimerism has shown promise and has been extended successfully to large animals and to the clinic; however, it remains cumbersome and requires heavy early immunosuppression. In this study, we reported that four injections of AMD3100, a CXCR4 antagonist, plus eight injections of low-dose FK506 (0.05 mg/kg per day) in the first week after kidney transplantation extended survival, but death from renal failure occurred at 30-90 days. Repeating the same course of AMD3100 and FK506 at 1, 2 and 3 mo after transplant resulted in 92% allograft acceptance (n = 12) at 7 mo, normal kidney function and histology with no further treatment. Transplant acceptance was associated with the influx of host stem cells, resulting in a hybrid kidney and a modulated host immune response. Confirmation of these results could initiate a paradigm shift in posttransplant therapy.

Cardiovascular safety of the dipetidyl peptidase-4 inhibitor alogliptin in type 2 diabetes mellitus.

AIM: As there have been concerns that some classes or agents for the treatment of type 2 diabetes may increase CV risk, we evaluated the cardiovascular profile of the dipeptidyl peptidase-4 inhibitor alogliptin. METHODS: We evaluated the incidence of CV events in patients treated with alogliptin, placebo or comparator antihyperglycaemic drugs in the clinical trial database for alogliptin using the composite major adverse cardiovascular event (MACE) endpoints of CV death, non-fatal myocardial infarction and non-fatal stroke. RESULTS: The pooled analysis included 4168 patients exposed to alogliptin 12.5 and 25 mg daily for 2023 patient-years compared to 691 patients treated with placebo for 263 patient-years and 1169 patients treated with other antidiabetic agents (metformin, sulfonylureas and thiazolidinediones) for 703 patient-years. CV events were adjudicated by an expert endpoint committee blinded to treatment allocation. The incidence rates of the combined MACE were not significantly different between patients treated with alogliptin and comparator therapies (hazard ratio=0.635, 95% confidence interval, 0.0, 1.41). Additionally, other types of serious CV events were not significantly different between patients treated with alogliptin and comparator therapies. CONCLUSION: These analyses have not shown a signal of increased CV risk with alogliptin in patients with type 2 diabetes. Future results from the adequately powered EXAMINE trial will definitively assess the CV safety profile of aloglipin in patients with type 2 diabetes mellitus.

Successful transplantation of reduced-sized rat alcoholic fatty livers made possible by mobilization of host stem cells.

Livers from Lewis rats fed with 7% alcohol for 5 weeks were used for transplantation. Reduced sized (50%) livers or whole livers were transplanted into normal DA recipients, which, in this strain combination, survive indefinitely when the donor has not been fed alcohol. However, none of the rats survived a whole fatty liver transplant while six of seven recipients of reduced sized alcoholic liver grafts survived long term. SDF-1 and HGF were significantly increased in reduced size liver grafts compared to whole liver grafts. Lineage-negative Thy-1+CXCR4+CD133+ stem cells were significantly increased in the peripheral blood and in allografts after reduced size fatty liver transplantation. In contrast, there were meager increases in cells reactive with anti Thy-1, CXCR4 and CD133 in peripheral blood and allografts in whole alcoholic liver recipients. The provision of plerixafor, a stem cell mobilizer, salvaged 5 of 10 whole fatty liver grafts. Conversely, blocking SDF-1 activity with neutralizing antibodies diminished stem cell recruitment and four of five reduced sized fatty liver recipients died. Thus chemokine insufficiency was associated with transplant failure of whole grafts, which was overcome by the increased regenerative requirements promoted by the small grafts and mediated by SDF-1 resulting in stem cell influx.

Mobilization of host stem cells enables long-term liver transplant acceptance in a strongly rejecting rat strain combination.

Careful examination of liver, kidney and heart transplants in human recipients has revealed small numbers of host bone marrow derived stem cells in the graft. If the limited recipient repopulation of a donor graft that is currently observed could be facilitated, it is possible that conversion to a predominantly host phenotype would permit long-term graft function without immunosuppression. We proposed to "engineer" repopulation after transplant in a strain combination (dark agouti [DA] to Lewis green fluorescent protein+[LEW GFP+]) which rejects liver grafts strongly, a model that more closely resembles the situation in humans. Treatment on days 0, 1, 2, 3 and 7 after transplantation with low-dose (0.1 mg/kg) tacrolimus (T) designed to blunt rejection combined with plerixafor (P) to mobilize host stem cells resulted in greater than 180 days graft survival with extensive albeit spotty conversion of a small (50%) DA graft to the recipient LEW GFP+ genotype. Subsequent skin grafting revealed donor-specific graft prolongation. The T plus P treatment resulted in higher levels of Lin-Thy1+CD34+CD133+ stem cells and Foxp3+ regulatory T cells in the blood and liver at day 7. Thus, pharmacological mobilization of host stem cells sustains liver allografts by two mechanisms: repopulation of injured donor cells and regulation of the immune response.

White blood cell counts in healthy Jamaican adults.

The investigation of presumed neutropenia places a burden on the health services, especially those of developing countries, including Jamaica. This may be because the normal ranges used in the laboratory are based on the values generated from the Caucasian population. Previous studies looking at African and Afro-Caribbean groups have found lower counts for these populations compared with Caucasians. To address this issue, 195 healthy adults donating blood at the National Public Health Laboratory and the University Hospital of the West Indies blood banks in Kingston, Jamaica, were screened for complete blood count (CBC) differentials between June 2001 and June 2006. The geometric means for the neutrophil counts were found to be 2.4 x 10(9)/L for men and 2.7 x 10(9)/L for women, with 95% confidence intervals of 2.2-2.8 x 10(9)/L and 2.5-3.1 x 10(9)/L respectively. Values for the Jamaican population were similar to those of other Afro-Caribbean groups. Based on this distribution, 14% of healthy Jamaicans would fall below the normal ranges derived from Caucasians and therefore presumed to have neutropenia. We recommend that the lower reference ranges obtained for Afro-Caribbean adults be adopted for that population.

Discrete serum protein signatures discriminate between human retrovirus-associated hematologic and neurologic disease.

The human T-cell leukemia virus type I (HTLV-I) is the causative agent for adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Approximately 5% of infected individuals will develop either disease and currently there are no diagnostic tools for early detection or accurate assessment of disease state. We have employed high-throughput expression profiling of serum proteins using mass spectrometry to identify protein expression patterns that can discern between disease states of HTLV-I-infected individuals. Our study group consisted of 42 ATL, 50 HAM/TSP, and 38 normal controls. Spectral peaks corresponding to peptide ions were generated from MS-TOF data. We applied Classification and Regression Tree analysis to build a decision algorithm, which achieved 77% correct classification rate across the three groups. A second cohort of 10 ATL, 10 HAM and 10 control samples was used to validate this result. Linear discriminate analysis was performed to verify and visualize class separation. Affinity and sizing chromatography coupled with tandem mass spectrometry was used to identify three peaks specifically overexpressed in ATL: an 11.7 kDa fragment of alpha trypsin inhibitor, and two contiguous fragments (19.9 and 11.9 kDa) of haproglobin-2. To the best of our knowledge, this is the first application of protein profiling to distinguish between two disease states resulting from a single infectious agent.

Multiple primary cancers in families with Li-Fraumeni syndrome.

BACKGROUND: Li-Fraumeni syndrome is a dominantly inherited disorder characterized by early-onset breast cancer, sarcomas, and other cancers in children and young adults. Members of families with this syndrome also develop multiple primary cancers, but the frequency is unknown. To approach this issue, we quantified the incidence of second and third primary cancers in individuals from 24 Li-Fraumeni kindreds originally diagnosed with cancer during the period from 1968 through 1986. METHODS: The relative risk (RR) of subsequent cancers and 95% confidence intervals (CIs) were calculated by use of population-based incidence data from the Connecticut Cancer Registry. Kaplan-Meier analysis was used to determine the cumulative probability (+/- standard error) of subsequent cancers. RESULTS: Among 200 Li-Fraumeni syndrome family members diagnosed with cancer, 30 (15%) developed a second cancer. Eight individuals (4%) had a third cancer, while four (2%) eventually developed a fourth cancer. Overall, the RR of occurrence of a second cancer was 5.3 (95% CI = 2.8-7.8), with a cumulative probability of second cancer occurrence of 57% (+/- 10%) at 30 years after diagnosis of a first cancer. RRs of second cancers occurring in families with this syndrome were 83.0 (95% CI = 36.9-187.6), 9.7 (95% CI = 4.9-19.2), and 1.5 (95% CI = 0.5-4.2) for individuals with a first cancer at ages 0-19 years, 20-44 years, and 45 years or more, respectively. Thirty (71%) of 42 subsequent cancers in this group were component cancers of Li-Fraumeni syndrome. CONCLUSIONS: Compared with the general population, members of Li-Fraumeni syndrome families have an exceptionally high risk of developing multiple primary cancers. The excess risk of additional primary cancers is mainly for cancers that are characteristic of Li-Fraumeni syndrome, with the highest risk observed for survivors of childhood cancers. Cancer survivors in these families should be closely monitored for early manifestations of new cancers.

Effects of glutathione depletion on the synthesis of proteoglycan and collagen in cultured chondrocytes.

We studied the effect of the depletion of glutathione on the synthesis of proteoglycan and collagen in cultured chick chondrocytes. When the cultured chondrocytes were incubated with 1 mM buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase, the intracellular glutathione level markedly dropped within 12 h with no loss of cell viability. Incorporation of 35SO2-4 into proteoglycan was lowered in the presence of BSO. When the 35S-labeled proteoglycans were separated into two fractions by glycerol density gradient centrifugation, the inhibitory effect of BSO on the synthesis of proteoglycan was greater in the fast-sedimenting proteoglycan fraction, which consisted mainly of cartilage specific large proteoglycan (PG-H), than in the slowly sedimenting proteoglycan fraction. The inhibition by BSO of the synthesis of core protein-free glycosaminoglycan chains primed by p-nitrophenyl-beta-D-xyloside was smaller than the inhibition of the synthesis of proteoglycan. Analysis of glycosaminoglycans labeled with [3H]glucosamine indicated that the treatment of chondrocytes with BSO resulted in a small increase in the proportion of synthesis of hyaluronic acid to the synthesis of total glycosaminoglycan. The incorporation of [3H]proline into collagen was also inhibited by BSO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the 3H-labeled collagen showed that, in the presence of BSO, processing of Type II collagen appeared to slow down and the proportion of Type X collagen synthesis was reduced.

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis.

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

Regional specialization in synaptic input and output in an identified local nonspiking interneuron of the crayfish revealed by light and electron microscopy.

Lateral inhibition of several mechanosensory interneurons in the crayfish terminal ganglion is mediated by a pair of identified local nonspiking interneurons called local directionally selective (LDS) interneurons. The ultrastructure and synaptic distribution of these interneurons were investigated by using intracellular labeling with horseradish peroxidase for electron microscopy. The LDS interneuron has bilateral, asymmetric arborizations. They are connected by a thick transverse neurite on which no synapses are made. The neurites on the side ipsilateral to the cell body are smooth and receive almost entirely input synapses. On the other hand, the contralateral neurites have prominent numerous swellings or varicosities from which many short fine spines arise, with intermingled input and output synapses. Both synapses, however, are not always associated with varicosities and spines. Presynaptic neurites of the LDS interneuron contain only round agranular synaptic vesicles in the vicinity of synaptic specializations. These morphological findings correlate well with the physiology of this interneuron: it is functionally polarized to receive input on one side (ipsilateral to the cell body) of the ganglion and convey it to the other side.

Distribution and ultrastructure of synapses on a premotor local nonspiking interneuron of the crayfish.

Many premotor local nonspiking interneurons are involved in the control of the uropod movements of a crayfish. One of these interneurons was impaled intracellularly, characterized physiologically, and then labeled by intracellular horseradish peroxidase (HRP) injection to examine the distribution and ultrastructure of synapses. Depolarization of this interneuron by a current injection excited the closer motoneurons of the uropod and inhibited its opener motoneurons, but hyperpolarization had no effect. Input and output synapses are distributed all over the major branches and the finer neurites except for the main neurite (7-10 micron in diameter), which runs near the dorsal surface of the neuropil. Both types of synapse are located on the same neurite and are often intermingled in close proximity, often less than 1 micron apart. Presynaptic terminals of the interneuron contain round, clear vesicles that are densely packed in fine branches and spines. The number of synaptic vesicles associated with a particular output synapse was estimated to be about 2,000. No dense-cored granules are observed in the labeled neurites. Our results support the proposal that synaptic transmission in this class of interneurons of the crayfish can sometimes be restricted to a very small region of the branches and that, therefore, different regions of the interneuron can function independently.

Opposing parallel connections through crayfish local nonspiking interneurons.

Unilateral local nonspiking interneurons in the terminal (sixth) abdominal ganglion of crayfish (Procambarus clarkii Girard) can be classified into two major groups of PL and AL types by their gross morphology and somatic position. These premotor interneurons are the neural components of uropod motor pattern formation. They receive sensory input from the exopodite of the contralateral side as well as that of the ipsilateral side. Small fluctuations in their membrane potentials cause sustained change in activity of the motoneurons innervating the uropod muscles. PL interneurons, which make noninverting connections to an identified closer, the reductor motoneuron No. 1, mainly receive excitatory input from the afferents of the contralateral exopodite, whereas inverting PL interneurons receive inhibitory input. AL interneurons receive distinctly different input from the afferents. Noninverting AL interneurons mainly receive inhibitory input, whereas inverting AL interneurons receive excitatory input. The rate of discharge of the reductor motoneurons is increased by sensory stimulation. The PL interneurons form either excitatory or disinhibitory pathways, which are relevant in function to the observed increase of the motoneuron. Conversely, the AL interneurons form either inhibitory or disfacilitatory pathways. Thus, the PL and the AL interneurons are fractionated in function and distinguishable in terms of their physiology by their input and output correlations. Functional meaning of the presence of these two types of unilateral local nonspiking interneurons of opposing connections in the uropod motor control system is discussed.

Intersegmental ascending interneurons controlling uropod movements of the crayfish Procambarus clarkii.

The premotor effects of intersegmental ascending interneurons upon uropod motor neurones in the crayfish Procambarus clarkii (Girard) are examined with intracellular recording and staining techniques. We show that many ascending interneurones can affect the activity of the antagonistic opener and closer motor neurones in the terminal ganglion. Based upon soma position, ascending interneurones are divided into three groups of rostral, medial, and caudal interneurones. Twenty-four ascending interneurones are characterized physiologically according to their inputs from the tailfan and their output effects on the uropod motor neurones of both sides. Each interneurone is identifiable as a unique individual by means of overall shape, soma position, number of main branches, the commissure in which primary neurites cross the midline, axon position in the 5th-6th abdominal connective and physiological responses. They are classified into six classes; coactivating, coinhibiting, reciprocally closing, reciprocally opening, variably effective, and not effective interneurones, according to their premotor effects on the uropod motor neurones. These ascending interneurones seem to act as multifunctional units conveying sensory information from the tailfan to the anterior abdominal ganglia and, at the same time, influencing the uropod motor pattern in the terminal abdominal ganglion.

Chronic immune thrombocytopenia in sarcoidosis.

We report a 69 year-old female with sarcoidosis who developed chronic thrombocytopenia, a rare complication of this disorder. Histologically normal bone marrow with increased number of megakaryocytes and high level of PAIgG strongly suggest the immune destruction of platelets as the cause of thrombocytopenia. In addition to thrombocytopenia, this case is also distinctive in its clinical manifestation. The patient developed several infrequent manifestations of sarcoidosis including complete AV block, uveitis, skin eruptions and middle lobe syndrome, but, did not have an intrathoracic adenopathy, the commonest manifestation of this disease.

Cardioactive peptides isolated from the brain of a Japanese octopus, Octopus minor.

Octopus cardioactive peptides (Ocp-1: Gly-D-Phe-Gly-Asp, and Ocp-3: Gly-Ser-Trp-Asp) were isolated from brain extracts of the octopus, Octopus minor, using the isolated systemic heart as a bioassay. These peptides showed both positive chronotropic and inotropic effects on the heart. The stereoisomers at position 2 were also isolated, but their activities were only 1/10(3)-1/10(4) those of the corresponding isomers. The presence of the peptides in the systemic heart was confirmed by time-of-flight mass spectrometry (MS) and tandem MS analysis. The results suggested that Ocp-1 and Ocp-3 might be involved in excitatory control of the octopus cardiovascular system as neuropeptides and/or neurohormones.

A novel GGNG-related neuropeptide from the polychaete Perinereis vancaurica.

The GGNG peptides are myoactive peptides so far identified from earthworms and leeches, which are the earthworm excitatory peptides (EEP) and the leech excitatory peptide (LEP), respectively. A novel GGNG peptide was isolated and structurally determined from a marine polychaete, Perinereis vancaurica, using a combination of immunological assay and high performance liquid chromatography (HPLC). The peptide was a pentadecapeptide whose amino acid sequence was similar to that of EEP and LEP, and showed myoactivity on isolated esophagus of P. vancaurica with a threshold concentration of 10(-10)M. The peptide was designated as polychaete excitatory peptide (PEP). Amidation of the alpha-carboxyl group of C-terminal residue occurred in PEP. This is the case for LEP, but not for EEP. The cDNA cloning revealed that the structure of the PEP precursor is more similar to the EEP precursor than to the LEP precursor. Immunohistochemical staining showed the presence of PEP in several neurons of central nervous system (CNS) as somata and neuropile structure, epithelial cells of the pharynx and epidermal cells throughout the body wall. Altogether these results support the physiological significance of PEP in regulation of the CNS neural activity and the peripheral myoactivity.

Tandem mass spectrometric analysis of (13)C-containing ions from a mixture of homologous peptides differing by one mass unit at a residue.

Tandem mass spectrometry of a mixture of two peptides that differ from each other by a single mass unit due to mutation is presented. The mutant beta-globin of hemoglobin Hoshida is present along with the normal counterpart, and the amino acid substitution of glutamine for glutamic acid is located within tryptic peptide T5 of M(r) 2057. 9. The mass of the mutated peptide is 1 u lower. In the isotopic cluster for the doubly charged ion of the peptide T5, the resolved ion with mass of 1030.0 represents the normal peptide with 93 (12)C atoms and the mutated one with 92 (12)C and one (13)C atoms. Collision-induced dissociation (CID) of this composite ion identified the mutation by presenting a key fragment derived from the (12)C-only mutant peptide, as reported in a previous study. Similarly, when an ion containing multiple (13)C atoms was selected as a precursor for CID, the mutation could be identified, even in large fragments, by a marked change in the shape of the isotopic cluster for the consecutive product ions. This study demonstrates the merit of selecting a resolved ion rather than the whole isotopic cluster as a precursor in the CID measurements of large peptides or proteins for characterizing heterozygous mutations.

Physiological and morphological characterization of anaxonic non-spiking interneurons in the crayfish motor control system.

Spike activation of the motoneurons innervating uropod muscles in crayfish is controlled by anaxonic interneurons located within the terminal (the 6th abdominal) ganglion. These neurons do not generate spikes either spontaneously at the resting potential level or in response to current injection of either polarity. Yet the change in the membrane potential of these non-spiking interneurons caused an increase or decrease in the discharge frequency of motoneuron spikes, depending upon the direction of the membrane potential change. These non-spiking interneurons within the terminal ganglion presumably integrate various descending command signals and select the adequate information to be gated to the motoneurons.

Alpha-pompilidotoxin (alpha-PMTX), a novel neurotoxin from the venom of a solitary wasp, facilitates transmission in the crustacean neuromuscular synapse.

A new neurotoxin, named alpha-pompilidotoxin (alpha-PMTX) has been found in the venom of the solitary wasp Anoplius safnariensis. In the neuromuscular synapse of the lobster walking leg preparation, alpha-PMTX (10-100 micro/M) caused great enhancement of both the excitatory and inhibitory postsynaptic potentials. Recordings of the excitatory post synaptic currents (EPSCs) at the synaptic sites showed that alpha-PMTX reversibly and dose-dependently potentiates EPSCs. Alpha-PMTX may act primarily on the presynaptic membrane but the mode of action of the toxin is clearly different from other known facilitatory neurotoxins, such as alpha-latrotoxin, apamin or charybdotoxin. This novel toxin will serve as a useful tool in the research field of neuroscience.

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins.

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation.