Inhibitory effects of the neurotensin<Superscript>8–13</Superscript> analogs Asp<Superscript>13</Superscript>-NT<Superscript>8–13</Superscript> and Asp<Superscript>12</Superscript>-NT<Superscript>8–13</Superscript> on mast cell secretion

Pretreatment of isolated mast cells with analogs of neurotensin 8–13 (NT^8–13), in which the amino acids Leu¹³ or Ile¹² are replaced with an aspartic acid (Asp¹³-NT^8–13 or Asp¹²-NT^8–13), inhibits the secretion of histamine in response to NT. A 10 min pretreatment with either analog (10 μM) inhibited NT-induced histamine release by 90% (Asp¹³-NT^8–13) or by 98% (Asp¹²-NT^8–13). At concentrations that are inhibitory, Asp¹³-NT^8–13 and Asp¹²-NT^8–13 alone elicit very little release (<5% at 10 μM). In the continued presence of the analogs, the inhibitory effect lasts for more than 45 min; removal of the analogs resulted in restoration of sensitivity to NT within 10 min. Pretreatment with analog Asp¹³-NT^8–13 resulted in a 39% inhibition of stimulation by substance P and a 52% inhibition of stimulation by histaminereleasing peptide (HRP). In contrast, pretreatment with analog Asp¹²-NT^8–13 gave no inhibition of release by SP or HRP. Neither analog inhibited histamine release in response to bradykinin (BK), NT^1–12, compound 48/80 (48/80), the calcium ionophore A23187, or anti-IgE stimulation of passively sensitized mast cells. Although Asp¹²-NT^8–13 and Asp¹³-NT^8–13 differ slightly in regard to the peptides they inhibit, both probably act at a step early in the stimulus-secretion coupling sequence; most likely before the rise in the level of free intracellular calcium that has been shown to accompany secretion in mast cells. It is suggested that these analogs exert their inhibitory effect on NT by competing with NT for a binding site on the mast cell membrane. The limited number of peptides inhibited by these analogs suggest that not all basic peptides act at the same site to stimulate secretion.     

Adenosine Triphosphoric Acid as a Factor of Nervous Regulation of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>/2Cl<Superscript>−</Superscript> Cotransport in Rat Skeletal Muscle Fibers

Exogenous adenosine triphosphoric acid produces a biphasic effect on the resting membrane potential of muscle fibers in rat diaphragm. Depolarization of the sarcolemma observed 10 min after application of adenosine triphosphoric acid results from activation of Na⁺/K⁺/2Cl⁻ cotransport. The increase in chloride cotransport is related to activation of postsynaptic P2Y receptors and protein kinase C. Repolarization of the membrane develops 40 min after treatment with adenosine triphosphoric acid and after 50 min the resting membrane potential almost returns the control level. This increase in the resting membrane potential of the sarcolemma is probably associated with activation of the Na⁺/K⁺ pump and increase in membrane permeability for chlorine ions in response to longterm activity of Cl⁻ cotransport. Thus, adenosine triphosphoric acid co-secreted with acetylcholine in the neuromuscular synapse probably plays a role in the regulation resting membrane potential and cell volume of muscle fibers.     

Interleukin-10 production and T cell-suppressive capacity in B cell subsets from atherosclerotic <Emphasis Type="Italic">apoE</Emphasis><Superscript><Emphasis Type="Italic">−/−</Emphasis></Superscript> mice

The evidence regarding the role of regulatory B cells (Breg) in atherosclerosis are scarce, and there are contradictory data about their atheroprotective properties. Due to the demonstrated protective function of Breg in different inflammatory diseases mainly through interleukin-10 (IL-10) production, the knowledge of their participation in atherosclerosis immunopathology would be very valuable. To further study which B cell subsets participate in IL-10 production and their regulatory role, splenocytes from apolipoprotein-E-deficient mice were evaluated by ex vivo and in vitro cultures. Atherosclerotic mice had increased frequency of IL-10⁺ B cells, which presented high CD1d, CD19, and IgM, but variable CD5, CD21, and CD23 expression. IL-10⁺ B cells were not enriched in B cell subsets previously reported as Breg. Increased frequency of IL-10⁺ B cells with transitional 1-like (T1-like) and follicular (FO) and reduced CD5⁺ and marginal zone (MZ) phenotypes were observed ex vivo. Increased frequency of IL-10⁺ B cells with T1-like and MZ, and decreased IL-10⁺ FO and T2 phenotypes were also observed in vitro. To determine regulatory capacity of B cells in the atherosclerotic model, each subset were co-cultured with CD4⁺CD25^? T cells. CD5⁺, FO, MZ, and T1-like cells from atherosclerotic mice exhibited regulation in an IL-10-dependent manner. However, only FO cells decreased both frequency of interferon gamma (IFN-γ)⁺ and tumor necrosis factor alpha (TNF-α)⁺ and proliferation of T cells. Finally, splenocytes showed increased frequency of IFN-γ⁺ and TNF-α⁺ cells only when FO-depleted B cells were evaluated. These results suggest that mainly FO B cells can modulate in some level the inflammatory responses observed in atherosclerosis.     

Chronotropic effect of D-Ala<Superscript>2</Superscript>,Leu<Superscript>5</Superscript>,Arg<Superscript>6</Superscript>-enkephalin (dalargin) is associated with activation of peripheral κ-opioid receptors

Intravenous infusion of D-Ala²,Leu⁵,Arg⁶-enkephalin (dalargin) caused bradycardia in narcotized rats. This effect was not observed during opioid receptor blockade with naloxone, naloxone methiodide, and norbinaltorphimine. Dalargin and (-)-U-50,488 added to Krebs—Henseleit perfusion solution for isolated rat heart decreased heart rate. Ganglionic blocker hexamethonium potentiated the negative chronotropic effect of dalargin. The negative chronotropic effect of dalargin is probably associated with activation of cardiac κ-opioid receptors. It should be noted that dalargin caused tachycardia in some animals. This reaction was not observed after treatment with hexamethonium. The positive chronotropic effect of dalargin is probably related to modulation of the parasympathetic autonomic nervous system. Agonists and antagonists of δ-opioid receptors caused persistent bradycardia. We hypothesized that selective δ-opioid antagonists exhibit properties of partial δ-receptor agonists. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 140, No. 12, pp. 633–638, December, 2005     

Brain inflammation is accompanied by peripheral inflammation in <Emphasis Type="Italic">Cstb</Emphasis><Superscript><Emphasis Type="Italic">−/−</Emphasis></Superscript> mice,a model for progressive myoclonus epilepsy

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited childhood-onset neurodegenerative disorder, characterized by myoclonus, seizures, and ataxia. Mutations in the cystatin B gene (CSTB) underlie EPM1. The CSTB-deficient (Cstb ^?/? ) mouse model recapitulates key features of EPM1, including myoclonic seizures. The mice show early microglial activation that precedes seizure onset and neuronal loss and leads to neuroinflammation. We here characterized the inflammatory phenotype of Cstb ^?/? mice in more detail. We found higher concentrations of chemokines and pro-inflammatory cytokines in the serum of Cstb ^?/? mice and higher CXCL13 expression in activated microglia in Cstb ^?/? compared to control mouse brains. The elevated chemokine levels were not accompanied by blood-brain barrier disruption, despite increased brain vascularization. Macrophages in the spleen and brain of Cstb ^?/? mice were predominantly pro-inflammatory. Taken together, these data show that CXCL13 expression is a hallmark of microglial activation in Cstb ^?/? mice and that the brain inflammation is linked to peripheral inflammatory changes, which might contribute to the disease pathology of EPM1.     

Maternal–neonatal erythrocyte membrane Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase and Mg<Superscript>2+</Superscript>-ATPase activities in relation to the mode of delivery

Free radical production and high catecholamine levels are implicated in the modulation of Na(+), K(+)-ATPase, and Mg(2+)-ATPase activities. The aim of this study was to investigate the effect of the mode of delivery on the above-mentioned enzyme activities in maternal-neonatal erythrocyte membrane. Women with normal pregnancy (N = 30) were divided into two groups: Group A (N = 16) with normal labor and vaginal delivery, and Group B (N = 14) with scheduled cesarean section; 20 non-pregnant women were the controls. Blood was obtained from controls and mothers, pre- versus post-delivery, and from the umbilical cord (CB). Total antioxidant status (TAS), membrane enzyme activities, and catecholamine blood levels were measured with a commercial kit, spectrophotometrically, and by HPLC methods, respectively. The results showed that: TAS levels, catecholamine, and the membrane enzyme activities were similar in the two groups of mothers pre-delivery, whereas both enzyme activities were lower than those of controls. TAS levels were reduced whereas Na(+), K(+)-ATPase activities (0.35 +/- 0.03 vs. 0.65 +/- 0.06 micromol Pi/h x mg protein, P < 0.001), and catecholamine levels were increased post-delivery in mothers of Group A and unaltered in Group B (0.38 +/- 0.02 vs. 0.40 +/- 0.03 micromol Pi/h x mg protein, P > 0.05), at the same times of study. Mg(2+)-ATPase activities remained unaltered in both groups of mothers and newborns. Na(+), K(+)-ATPase activity was similarly lower in the CB of neonates than those of their mothers, pre-delivery. Our results suggest that: (a) during a normal vaginal delivery process, the low TAS and the increased levels of catecholamines may increase Na(+), K(+)-ATPase activity, post-delivery; (b) the low enzyme activities evaluated in mothers pre-delivery may be due to the high estrogen levels and those in newborns due to perinatal immaturity.     

Effect of interleukin-1β on NMDA-induced <Superscript>45</Superscript>Ca<Superscript>2+</Superscript> uptake by synaptosomes of rat brain cortex

The effect of interleukin-1β on presynaptic NMDA receptors was evaluated by studying NMDA-induced ⁴⁵Ca²⁺ uptake by synaptosomes from rat brain cortex. Interleukin-1β inhibited ⁴⁵Ca²⁺ uptake by synaptosomes. Our results indicate that interleukin-1β modulates presynaptic NMDA receptors and is probably involved in the regulation of synaptic transmission in the central nervous system. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 140, No. 12, pp. 645–646, December, 2005     

Apoptosis Inhibitor Expressed by Macrophages Tempers Autoimmune Colitis and the Risk of Colitis-Based Carcinogenesis in TCRα<Superscript>−/−</Superscript> Mice

Background Crohn’s disease and ulcerative colitis (UC) are the two main entities involved in human inflammatory bowel disease (IBD). However, their precise etiologies remain unclear. To study the development of mucosal inflammation, and chronic inflammation-based dysplasia and carcinoma formation, we examined possible roles of the apoptosis inhibitor expressed by macrophages (AIM) in an experimental IBD model. Methods In this study, we used T cell receptor α deficient (TCRα^−/−) mice, a known UC-like colitis model. We generated TCRα^−/− × AIM^−/− double knockout mice by crossbreeding TCRα^−/− with AIM^−/− mice. At 24 weeks of age, mice were killed to obtain colon tissues for pathological examinations. TCRα^−/− × AIM^+/− mice, heterozygous littermates of TCRα^−/− × AIM^−/− mice, were used as controls. Results Severe colitis was observed in TCRα^−/− × AIM^−/− mice, when compared with TCRα^−/− × AIM^+/− mice. Dysplasia was detected in TCRα^−/− × AIM^−/− mice, but not in TCRα^−/− × AIM^+/− mice. Adenocarcinoma formation was observed from dysplasia only in TCRα^−/− × AIM^−/− mice. Conclusion Not only a high incidence of severe colitis but also dysplasia and adenocarcinoma formation were observed in TCRα^−/− × AIM^−/− mice only. AIM have some regulatory roles in inflammation and progression of dysplasia to carcinoma in TCRα^−/− mice.     

Use of a <Emphasis Type="Italic">ura5</Emphasis><Superscript>+</Superscript>–<Emphasis Type="Italic">lys7</Emphasis><Superscript>+</Superscript> cassette to construct unmarked gene knock-ins in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

While the counterselectable Schizosaccharomyces pombe ura4 ⁺ gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4 ⁺ confers 5FOA-resistant (5FOA^R) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA^R mutants. Relative to the same approach using the homologous URA3 ⁺ gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower efficiency of homologous recombination and a relatively high background of spontaneous 5FOA^R colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we first determined that 5FOA^R strains carry mutations in either of two genes; ura4 ⁺ and ura5 ⁺. We then cloned the S. pombe ura5 ⁺ orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5 ⁺ together with the S. pombe lys7 ⁺ gene. Using this doubly marked cassette to disrupt the sck1 ⁺ kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4 ⁻ and ura5 ⁻ mutants by screening 5FOA^R colonies for the loss of the lys7 ⁺ marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that ~95% of 5FOA^R colonies in our studies arose from background ura4 ⁻ and ura5 ⁻ mutations.     

In vitro binding evaluation of <Superscript>177</Superscript>Lu-AMBA,a novel <Superscript>177</Superscript>Lu-labeled GRP-R agonist for systemic radiotherapy in human tissues

Members of the gastrin-releasing peptide (GRP) family and its analogs bombesin (BBN) have been implicated in the biology of several human cancers including prostate, breast, colon and lung. To date, three mammalian GRP/BBN receptor subtypes have been cloned and characterized: the neuromedin B receptor (NMBR), the GRP receptor (GRPR) and the BBN-receptor subtype 3 (BB₃). The fourth BBN receptor subtype, BB₄, has only been identified in amphibian and at present no mammalian equivalent of this receptor has been described. GRPR analogs have been used as carriers to deliver drugs, radionuclides and cytotoxins to target various cancer types that are GRPR positive. We investigated the in vitro binding properties of ¹⁷⁷Lu-AMBA, a novel radiolabelled BBN analog currently undergoing clinical trial as systemic radiotherapy for hormone refractory prostate cancer (HRPC) patients. Pharmacological analyses of the ¹⁷⁷Lu-AMBA was determined using in vitro binding studies using membrane target system containing specific receptor subtypes. We investigated the distribution of binding sites for ¹⁷⁷Lu-AMBA by receptor autoradiography on human neoplastic and non-neoplastic tissues. Pharmacological characterizations of ¹⁷⁷Lu-AMBA shows, high affinity towards NMB and GRP receptors, while little or no affinity towards BB₃ receptor. Among the 40 different types of non-neoplastic tissues tested seven of them showed limited but specific binding of ¹⁷⁷Lu-AMBA. Fourteen of 17 primary prostate cancers, six of 13 primary breast cancers expressed binding sites for ¹⁷⁷Lu-AMBA. Furthermore, no apparent differences in ¹⁷⁷Lu-AMBA-binding sites expression were observed between matched pairs (primary vs. secondary) of prostate and breast cancer tissues. These data represent the molecular basis for clinical applications of ¹⁷⁷Lu-AMBA for diagnosis and treatment of GRP-R and NMB-R positive tumors.     

Sorghum stem extract modulates Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase,ecto-5′-nucleotidase,and acetylcholinesterase activities

Sorghum stem (Sorghum bicolor) has been in use in traditional medicine systems for the management of neurodegenerative conditions. However, there is dearth of information on the scientific basis for its use in the treatment of such conditions. This study sought to assess the antioxidant activity and effects of phenolic extract from sorghum stem (Sorghum bicolor) on some cholinergic (acetylcholinesterase (AChE)) and purinergic (Na⁺/K⁺-ATPase and ecto-5′-nucleotidase) enzymes associated with neurological conditions. Phenolic-rich extract was prepared using methanol: 1 N HCl (1:1, v/v) mixture and characterized using high-performance liquid chromatography-diode array detector (HPLC-DAD). In vitro tests were used to investigate the effects of the phenolic extract on AChE, Na⁺/K⁺-ATPase, and ecto-5′-nucleotidase activities. Furthermore, the hydroxyl (OH) radical scavenging and Fe²⁺-chelating abilities of the extract were investigated. HPLC-DAD analysis revealed the presence of some phenolic acids such as caffeic acid (120.58 mg/g), ferulic acid (76.45 mg/g), gallic acid (17.48 mg/g), chlorogenic acid (16.25 mg/g), and flavonoids such as kaempferol (15.98 mg/g), rutin (51.07 mg/g), quercetin (263.16 mg/g), and quercitrin (89.21 mg/g) in the phenolic extract. The extract significantly inhibited AChE and ecto-5′-nucleotidase activities in a dose dependent manner with IC₅₀?=?24.88 μg/ml and IC₅₀?=?37.49 μg/ml, respectively, and increased Na⁺/K⁺-ATPase activity in a dose dependent manner. Furthermore, the phenolic extract also scavenged OH radicals and was able to chelate Fe²⁺ in a dose dependent manner with IC₅₀?=?54.27 μg/ml and 18.47 μg/ml, respectively. This study revealed the antioxidant and modulatory effects of phenolic extracts from sorghum stem on some cholinergic and purinergic enzymes.     

Platelet deficiency in <Emphasis Type="Italic">Tpo</Emphasis><Superscript>−/−</Superscript> mice can both promote and suppress the metastasis of experimental breast tumors in an organ-specific manner

Platelets are thought to play an important role in metastasis formation, although the mechanisms involved remain incompletely understood. Here we studied the influence of platelet numbers on organ-specific metastasis to the lungs and lymph nodes using Tpo deficient mice that have low platelet counts. After tail vein injection of 4T1 breast cancer cells, the number of lung metastases was significantly lower in Tpo^?/? mice compared to Tpo^+/+ mice. The same was true for the bone-tropic 4T1.2 derivative. In spontaneous orthotopic metastasis assays, 4T1 and 4T1.2 primary tumor growth was not affected by the genotype of the mice. However, the number of 4T1.2 lung metastases was significantly lower in Tpo^?/? mice compared to Tpo^+/+ mice, whereas the number of 4T1 lung metastases was unaffected. Moreover, in mice bearing 4T1 tumors, lymph node metastases were larger in the Tpo^?/? background, and lymph node metastasis frequency was higher in Tpo^?/? mice bearing 4T1.2 tumors compared to that in wild-type mice. Enhanced lymph node metastasis in Tpo^?/? mice was not associated with changes in peritumoral lymphatic vessel density in the primary tumors. Together, our data indicate that platelets do not affect primary tumor growth in this breast cancer model, but can differentially influence site-specific metastasis to lymph nodes and lungs.     

Extraction of fetal electrocardiogram using <Emphasis Type="Italic">H</Emphasis><Superscript>∞</Superscript> adaptive algorithms

The fetal electrocardiogram (fECG) contains important information regarding the health of the fetus. However, the fECG obtained noninvasively from the abdominal surface electrical recordings of a pregnant woman are dominated by strong interference from the maternal electrocardiogram (mECG). In this paper, based on the H(infinity) principle, two adaptive algorithms are proposed for the extraction of fECG from the trans-abdominal recordings of pregnant women. The motivation behind the application of H(infinity) techniques is the fact that they are robust with respect to model uncertainties and lack of statistical information regarding noise. The proposed algorithms are applied to simulated as well as real multichannel ECG recordings and their performances are compared to that of the well-known least-mean-square (LMS) adaptive algorithm. It is found that the proposed H(infinity) based algorithms perform superior to the LMS algorithm in extracting the fECG signal.     

HCO<Subscript>3</Subscript><Superscript>−</Superscript>-dependent transient acidification induced by ionomycin in rat submandibular acinar cells

Ionomycin (IM, 5 μM), which exchanges 1 Ca²⁺ for 1 H⁺, changed intracellular pH (pH_i) with Ca²⁺ entry into rat submandibular acinar cells. IM-induced changes in pH_i consisted of two components: the first is an HCO₃ ⁻-dependent transient pH_i decrease, and the second is an HCO₃ ⁻-independent gradual pH_i increase. IM (1 μM), which activates store-operated Ca²⁺ channels, induced an HCO₃ ⁻-dependent and transient pH_i decrease without any HCO₃ ⁻-independent pH_i increase. Thus, a gradual pH_i increase was induced by the Ca²⁺/H⁺ exchange. The HCO₃ ⁻-dependent and transient pH_i decrease induced by IM was abolished by acetazolamide, but not by methyl isobutyl amiloride (MIA) or diisothiocyanatostilbene disulfonate (DIDS), suggesting that the Na⁺/H⁺ exchange, the Cl⁻/HCO₃ ⁻ exchange, or the Na⁺-HCO₃ ⁻ cotransport induces no transient pH_i decrease. Thapsigargin induced no transient pH_i decrease. Thus, IM, not Ca²⁺ entry, reduced pH_i transiently. IM reacts with Ca²⁺ to produce H⁺ in the presence of \textCO ₂ /\textHCO ₃ ^- :  [ \textH - \textIM ] ^- + \text Ca ²⁺  + \textCO ₂ \rightleftarrows [ \textH-\textCa - \textIM ] ⁺ ·\textHCO ₃ ^- + \textH ⁺ {\text{CO}}_{ 2} /{\text{HCO}}_{ 3}{^{ - }} : \, \left[ {{\text{H}} - {\text{IM}}} \right]^{ - } + {\text{ Ca}}^{ 2+ } \,+ {\text{CO}}_{ 2} \rightleftarrows \left[ {{\text{H}}-{\text{Ca}} - {\text{IM}}} \right]^{ + } \cdot {\text{HCO}}_{ 3}{^{ - } }+ {\text{H}}^{ + } . In this reaction, a monoprotonated IM reacts with Ca²⁺ and CO₂ to produce an electroneutral IM complex and H⁺, and then H⁺ is removed from the cells via CO₂ production. Thus, IM transiently decreased pH_i. In conclusion, in rat submandibular acinar cells IM (5 μM) transiently reduces pH_i because of its chemical characteristics, with HCO₃ ⁻ dependence, and increases pH_i by exchanging Ca²⁺ for H⁺, which is independent of HCO₃ ⁻.     

Increased O<Subscript>2</Subscript> consumption in excitation–contraction coupling in hypertrophied rat heart slices related to increased Na<Superscript>+</Superscript>–Ca<Superscript>2+</Superscript> exchange activity

The goal of our study was to evaluate the origin of the increased O₂ consumption in electrically stimulated left ventricular slices of isoproterenol-induced hypertrophied rat hearts with normal left ventricular pressure. O₂ consumption per minute (mVO₂) of mechanically unloaded left ventricular slices was measured in the absence and presence of 1-Hz field stimulation. Basal metabolic mVO₂, i.e., mVO₂ without electrical stimulation, was significantly smaller, but mVO₂ for the total Ca²⁺ handling in excitation–contraction coupling (E–C coupling mVO₂), i.e., delta mVO₂ (=mVO₂ with stimulation − mVO₂ without stimulation), was significantly larger in the hypertrophied heart. Furthermore, the fraction of E–C coupling mVO₂ was markedly altered in the hypertrophied heart. Namely, mVO₂ consumed by sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) was depressed by 40%; mVO₂ consumed by the Na⁺/K⁺-ATPase (NKA)-Na⁺/Ca²⁺ exchange (NCX) coupling was increased by 100%. The depressed mVO₂ consumption by SERCA2 was supported by lower protein expressions of phosphorylated-Ser¹⁶ phospholamban and SERCA2. The increase in NKA–NCX coupling mVO₂ was supported by marked augmentation of NCX current. However, the increase in NCX current was not due to the increase in NCX1 protein expression, but was attributable to attenuation of the intrinsic inactivation mechanisms. The present results demonstrated that the altered origin of the increased E–C coupling mVO₂ in hypertrophy was derived from decreased SERCA2 activity (1ATP: 2Ca²⁺) and increased NCX activity coupled to NKA activity (1ATP: Ca²⁺). Taken together, we conclude that the energetically less efficient Ca²⁺ extrusion pathway evenly contributes to Ca²⁺ handling in E–C coupling in the present hypertrophy model.     

T<Superscript>+</Superscript> NK<Superscript>+</Superscript> IL-2 Receptor γ Chain Mutation: a Challenging Diagnosis of Atypical Severe Combined Immunodeficiency

Purpose

All reported patients with hypomorphic X-linked severe combined immunodeficiency (X-SCID) due to c.664C>T (p.R222C) mutations in the gene (IL2RG) encoding the common γ chain (γc) have presented with opportunistic infections within the first year of life, despite the presence of nearly normal NK and T cell numbers. Reporting five children of one extended family with hemizygous mutations in IL2RG, we explore potential diagnostic clues and extend our comprehension of the functional impact of this mutation.

Methods

Whole exome sequencing (WES); detailed immune phenotyping; cytokine-induced STAT phosphorylation; B, T, and NK cell activation; and quantification of sjTRECs in five Arab children with c.664C>T (p.R222C) IL2RG mutation.

Results

The mean age at clinical presentation with respiratory tract infection or diarrhea was 6.8 (range: 2–12) months. None of the children presented with opportunistic infections. Diagnostic clues were early onset in the first year of life, and a suggestive family history associated with reduced naïve CD4 T cells and absent switched memory B cells. Number and phenotype of NK cells and innate-like lymphocytes were normal. The diagnosis was made by WES and corroborated by absent STAT phosphorylation and reduced functional response after IL-2 and IL-21 stimulation. Four patients underwent successful hematopoietic stem cell transplantation.

Conclusions

As early diagnosis and treatment are important, a high index of suspicion in the diagnosis of c.664C>T (p.R222C) X-SCID is needed. This requires prompt genetic testing by next generation sequencing in order to avoid unnecessary delays in the definite diagnosis since immunological work up may not be discriminating. Assays directly testing cytokine signaling or cytokine-dependent functions are helpful in confirming the functional impact of the identified hypomorphic variants.

     

Association of HLA DQ B1<Superscript>*</Superscript> and HLA DR B1<Superscript>*</Superscript> Alleles with Goitrous Juvenile Autoimmune Hypothyroidism—A Case Control Study

The present study was designed to analyze the distribution of HLA DQ B1* and DR B1* in patients with goitrous juvenile autoimmune hypothyroidism from Hyderabad, India. Analysis indicated an increase in the frequencies of HLA DQ B1* 03 allele (P < 0.000) and HLA DR B1* 04 (P < 0.05) alleles in this group of patients when compared to the controls, whereas the frequency of DQB1* 05 was found to be decreased in patients' group compared to the controls (p < 0.01). To conclude, we report a positive correlation between DQ B1* 03 and DR B1* 04 and goitrous juvenile autoimmune hypothyroidism, whereas DQB1* 05 is observed to be negatively correlated with this thyroid dysfunction. Since the disease-susceptible HLA class II alleles appear to differ in various ethnic groups for some autoimmune diseases, the observed association from Indian series of patients holds significance.     

Effects of<Emphasis Type="Italic"> Eimeria separata</Emphasis> infections on Na<Superscript>+</Superscript> and Cl<Superscript>−</Superscript> transport in the rat large intestine

To study the pathophysiology of diarrhoea in coccidial infections, Na⁺ and Cl^– fluxes (J_Na, J_Cl), short circuit current (I_sc) and tissue conductance (g_t) were determined in stripped gut epithelia of Eimeria separata infected rats employing the Ussing chamber technique. E. separata invades enterocytes of the caecum and proximal colon. Na⁺ absorption was generally reduced in infected tissues, Cl^– absorption only in the caecum. I_sc values were increased in the caecum and reduced in the proximal colon. Tissue conductance was not affected. Values tended to normal with time after infection. Theophylline caused markedly increased I_sc and g_t values in the caecum epithelia of infected rats. In the epithelia of the distal colon, i.e. the non-infested part of the large intestine, g_t values remained unaffected but I_sc was fourfold increased. This I_sc increase was strongly sensitive to amiloride, suggesting a compensatory activation of Na⁺ channels in the distal colon of infected rats. Accordingly, serum levels of aldosterone, which activates Na⁺ channels in the distal colon, were increased to eightfold levels in infected animals. Thus compensatory Na⁺ absorption was under endocrine control.     

Effect of H<Subscript>1</Subscript>-antagonist Dithiaden<Superscript>®</Superscript> on human PMN-leukocyte aggregation and chemiluminescence is stimulus-dependent

OBJECTIVE AND DESIGN: Contradictory data published on histamine-PMN leukocyte interactions stimulated us to study to the role of histamine and H1-antagonist Dithiaden in generation of reactive oxygen species (ROS) and aggregation of human neutrophils. METHODS AND MATERIALS: Whole blood or isolated PMN-leukocytes were exposed in a dose-dependent way to histamine or H1-antagonist Dithiaden and subsequently stimulated. Whole blood was stimulated with opsonised zymosan (OZ). Isolated cells were stimulated with membrane stimuli (OZ, N-formyl-methionyl-leucyl-phenylalanine--fMLP), or membrane bypassing stimuli (Ca2+-ionophore A23187, phorbol-myristate-acetate--PMA). The luminol-enhanced chemiluminescence (CL) was measured separately (whole blood) in a luminometer or simultaneously with neutrophil aggregation in a whole blood lumiaggregometer. RESULTS: Depending on the concentration used, Dithiaden" was 1.5- to 25.0-times more effective in inhibiting activated CL of whole blood than histamine. In isolated neutrophils both histamine and Dithiaden inhibited OZ- and A23187-stimulated CL dose-dependently, with potentiation observed after stimulation with PMA and fMLP. Histamine did not alter aggregation with any of the stimuli tested. Dithiaden inhibited A23187-, OZ- and PMA-stimulated PMN-leukocytes but potentiated fMLP-induced aggregation of isolated neutrophils. Simultaneous application of Dithiaden and histamine abolished the effect of Dithiaden on fMLP-stimulated CL. CONCLUSIONS: Dithiaden, depending on the stimuli applied, inhibited human neutrophils, both isolated or in whole blood, more markedly than histamine. The inhibition of aggregation and CL was dose- and stimulus-dependent. Histamine administered simultaneously abolished the effect of Dithiaden on fMLP-stimulated PMN-leukocytes. It seems likely that the interaction of Dithiaden with neutrophils operated both at an extra- and intracellular level.