Effects of recombinant cytokines on murine megakaryocyte colony formation in a serum-free fibrin clot culture system.

A convenient serum-free fibrin clot culture system for murine megakaryocyte progenitor cells was developed. The culture and counting of colonies is much easier in this system, when compared with previously reported serum-free culture methods. Recombinant murine interleukin-3 (rmIL-3) stimulated megakaryocyte colony formation in a dose-dependent manner in this system. While recombinant human granulocyte colony-stimulating factor (rhG-CSF) had no effect on megakaryocytopoiesis, recombinant human erythropoietin (rhEpo) and recombinant human interleukin-6 (rhIL-6) augmented megakaryocyte colony formation stimulated by rmIL-3. The depletion of adherent cells and T cells from the cultured bone marrow did not eliminate the synergistic effect of rhEpo and rhIL-6.     

Multispacer typing of Rickettsia prowazekii enabling epidemiological studies of epidemic typhus

Currently, there is no tool for typing Rickettsia prowazekii, the causative agent of epidemic typhus, currently considered a potential bioterrorism agent, at the strain level. To test if the multispacer typing (MST) method could differentiate strains of R. prowazekii, we amplified and sequenced the 25 most variable intergenic spacers between the R. prowazekii and R. conorii genomes in five strains and 10 body louse amplicons of R. prowazekii from various geographic origins. Two intergenic spacers, i.e., rpmE/tRNA(fMet) and serS/virB4, were variable among tested R. prowazekii isolates and allowed identification of three and two genotypes, respectively. When the genotypes obtained from the two spacers were combined, we identified four different genotypes. MST demonstrated that several R. prowazekii strains circulated in human body lice during an outbreak of epidemic typhus in Burundi. This may help to discriminate between natural and intentional outbreaks. Our study supports the usefulness of MST as a versatile method for rickettsial strain genotyping.     

The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues

The human non-classical MHC class I molecule HLA-E is a ligand for both an inhibitory NK cell receptor (CD94/NKG2A) and an activating receptor (CD94/NKG2C). To identify HLA-E surface recognized by both receptors, especially to determine if both receptors recognize the same epitope, we made a series of individually Ala-substituted HLA-E proteins and analyzed their binding to CD94/NKG2A orCD94/NKG2C. Eight HLA-E mutations that significantly impaired HLA-E binding to CD94/NKG2A are all found in the top of alpha1/alpha2 domain of HLA-E. These results suggest that CD94/NKG2A binds a HLA-E surface equivalent to a NKG2D binding site on MICA. Of the eight mutations that impaired HLA-E binding to CD94/NKG2A, six significantly impaired HLA-E binding to CD94/NKG2C suggesting that CD94/NKG2C also binds a similar surface of HLA-E. Unexpectedly, the two HLA-E mutations (D69A and H155A) selectively abrogated HLA-E binding to CD94/NKG2A, not largely affected CD94/NKG2C. These results indicate that a mostly shared, but partly distinct set of HLA-E residues is discriminated by the two receptors.     

The Serum Factor from Patients with Ulcerative Colitis that Induces T Cell Proliferation in the Mouse Thymus Is Interleukin-7

The disturbance of immune regulatory T cells is related to the pathogenesis of ulcerative colitis. Here we demonstrated and characterized the serum factor from ulcerative colitis patients that induced proliferation of intrathymic T cells. The factor isolated from the patient sera by a combination of gel filtration and anion-exchange chromatography induced proliferation of CD4⁺CD8^– intrathymic T cells in the organ-cultured embryonic mouse thymus. Purification and amino acid sequence analysis of the serum factor demonstrated that the N-terminal 12 sequence was homologous to that of interleukin-7. SDS-PAGE and Western blot confirmed that purified serum factor was interleukin-7. Enzyme immunoassay demonstrated that the serum interleukin-7 concentration was significantly increased in the patients. PCR and Southern blot hybridization demonstrated that interleukin-7 mRNA expression was increased in the thymus tissues from patients but decreased in the colonic mucosa. Since interleukin-7 is a crucial cytokine for proliferation and differentiation of T cells in the thymus, the present study indicates that interleukin-7 may contribute to the disturbance of immune regulatory T cells in ulcerative colitis.     

On the various manifestations of spontaneous autoimmune diabetes in rodent models

Autoimmune (type 1) diabetes mellitus in mouse, rat, and humans shares several features, including T lymphocyte infiltration into pancreatic islets and a dependence on permissive class II major histocompatibility complex (MHC) alleles. We report here on an experimental model involving mice that express influenza hemagglutinin (HA) under the control of the insulin promoter and, at the same time, a transgenic class II MHC-restricted T cell receptor (TcR) specific for an HA peptide. These mice spontaneously develop islet infiltrates resembling those found in NOD mice and most animals become diabetic within 8 weeks of age. Because of the availability of a clonotypic TcR antibody, we can be confident that the Ins-HA transgene does not induce any measurable alterations in the vast majority of T cells with the transgenic TcR in primary and secondary lymphoid organs. Continuous export of large numbers of HA-specific lymphocytes from the thymus was not required for the manifestation of the disease since mice thymectomized at 3 days after birth still developed the disease albeit with smaller infiltrates.     

The relationships between autonomic function and heart rate in essential hypertension

The circadian variation of blood pressure (BP) may be mediated at least in part by circadian variation of autonomic nervous system activity. In the present study power spectral analysis of hourly R-R intervals for 24 hours was done to obtain the low frequency variability (LF: 0.04 to 0.15Hz) and high frequency variability(HF: 0.15 to 0.40Hz) in 93 patients with untreated essential hypertension. LF/HF ratio and HF were considered to be an index of sympathetic nervous activity and parasympathetic nervous activity, respectively. The relationship between heart rate (HR) and systolic blood pressure (SBP), diastolic blood pressure (DBP), HF, and LF/HF was examined. Both SBP and DBP were correlated positively with HR(r = 0.28, p < 0.05 and r = 0.27, p < 0.05, respectively) in the daytime. HF was correlated negatively and significantly with HR in both daytime and nighttime (r = -0.40, p < 0.05, and r = -0.38, p < 0.05 respectively). After averaging for SBP of 24 hours, HR in the patients whose SBP was 135mmHg or more was significantly higher than that in the patients whose SBP was less than 135mmHg. These results suggest that the decreased parasympathetic nervous activity seems to be responsible for the rise in BP.     

Long‐term observation of a Japanese mucolipidosis IV patient with a novel homozygous p.F313del variant of MCOLN1

Mucolipidosis type IV (MLIV) is an autosomal recessively inherited lysosomal storage disorder characterized by progressive psychomotor delay and retinal degeneration that is associated with biallelic variants in the MCOLN1 gene. The gene, which is expressed in late endosomes and lysosomes of various tissue cells, encodes the transient receptor potential channel mucolipin 1 consisting of six transmembrane domains. Here, we described 14‐year follow‐up observation of a 4‐year‐old Japanese male MLIV patient with a novel homozygous in‐frame deletion variant p.(F313del), which was identified by whole‐exome sequencing analysis. Neurological examination revealed progressive psychomotor delay, and atrophy of the corpus callosum and cerebellum was observed on brain magnetic resonance images. Ophthalmologically, corneal clouding has remained unchanged during the follow‐up period, whereas optic nerve pallor and retinal degenerative changes exhibited progressive disease courses. Light‐adapted electroretinography was non‐recordable. Transmission electron microscopy of granulocytes revealed characteristic concentric multiple lamellar structures and an electron‐dense inclusion in lysosomes. The in‐frame deletion variant was located within the second transmembrane domain, which is of putative functional importance for channel properties.     

Effects of growth hormone treatment on thyroid function in pediatric patients with Prader–Willi syndrome

It is unclear whether hypothyroidism is present in patients with Prader–Willi syndrome (PWS). This study aimed to clarify the state of the hypothalamic–pituitary–thyroid axis and the effects of growth hormone (GH) treatment on thyroid function in pediatric patients with PWS. We retrospectively evaluated thyroid function in 51 patients with PWS before GH treatment using a thyroid‐releasing hormone (TRH) stimulation test (29 males and 22 females; median age, 22 months). We also evaluated the effect of GH therapy on thyroid function by comparing serum free triiodothyronine (fT3), free thyroxine (fT4), and thyroid stimulating hormone (TSH) levels at baseline, 1 year, and 2 years after GH therapy. TSH, fT4, and fT3 levels were 2.28 μU/ml (interquartile range [IQR]; 1.19–3.61), 1.18 ng/dl (IQR; 1.02–1.24), and 4.02 pg/dl (IQR; 3.54–4.40) at baseline, respectively. In 49 of 51 patients, the TSH response to TRH administration showed a physiologically normal pattern; in two patients (4.0%), the pattern suggested hypothalamic hypothyroidism (delayed and prolonged TSH peak after TRH administration). TSH, fT4, and fT3 levels did not change significantly during 1 or 2 years after GH treatment. The TSH response to TRH showed a normal pattern in most patients, and thyroid function did not change significantly during the 2 years after initiating GH treatment.     

Experimental autoimmune thyroiditis in nonobese diabetic mice lacking interferon regulatory factor-1

Interferon regulatory factor-1 (IRF-1) is pivotal in the regulation of interferon (IFN)-mediated immune reactions, and studies suggest that IRF-1 is involved in the development of autoimmune diseases. IRF-1+/+, +/-, and -/- nonobese diabetic (NOD) mice were immunized with mouse thyroglobulin (mTg) to determine whether IRF-1 is required in experimental autoimmune thyroiditis (EAT), a murine model for Hashimoto's thyroiditis (HT). IRF-1-deficient mice developed EAT and anti-mTg antibodies comparable to IRF-1+/+ and +/- mice. Whereas both CD4+ and CD8+ T cells were found in thyroids of IRF-1+/+ mice, the latter was not in IRF-1-/- mice. Major histocompatibility complex class II antigen was comparably expressed in thyroids of IRF-1+/+ and -/- mice. Lack of IRF-1 resulted in decreased CD8+ T cell number in the spleen and reduced IFNgamma production by splenocytes. Our results suggest that IRF-1 is not pivotal in EAT in NOD mice.     

Depolarization thresholds for hippocampal damage, ischemic preconditioning, and changes in gene expression after global ischemia in the rat

Induced ischemic tolerance in rat hippocampus was investigated in a forebrain ischemia model of repeated 4-vessel occlusion (4-VO). Ischemic insult variability was reduced by the use of dc potential measurements to determine the duration of ischemic depolarization in hippocampus. The results demonstrate a depolarization threshold for ischemic injury to CA1 neurons of 4-6 min and a window for optimal preconditioning of 2.5-3.5 min. Levels of induced mRNAs encoding hsp72 and several immediate-early genes were also shown to vary with depolarization interval. Immediate-early genes were maximally induced after depolarization periods inducing optimal preconditioning, while hsp72 expression increased with insult severity over the range leading to neuron loss. These results are similar to those obtained in gerbil studies indicating that preconditioning does not require large increases in hsp72 expression, and demonstrate the fundamental comparability of rodent global ischemia models when monitored by this approach.