ID Mice Research Articles

Dietary Iron Deficiency Modulates Adipocyte Iron Homeostasis, Adaptive Thermogenesis, and Obesity in C57BL/6 Mice

BackgroundAdaptive thermogenesis is an iron-demanding pathway, significantly contributing to whole-body energy expenditure. However, the effects of iron-deficient diets on adaptive thermogenesis and obesity remain unknown. ObjectivesWe aimed to determine the impact of dietary iron deficiency on iron homeostasis in adipocytes, adaptive thermogenic capacity, and metabolic consequences in obesity. MethodsC57BL/6 male mice were assigned to either the iron-adequate (IA, 35 ppm) or the iron-deficient group (ID, 3 ppm) at weaning. Upon 8 wk of age, both IA and ID groups received an isocaloric high-fat diet (45% kcal from fat) for 10 wk, maintaining the same iron content. Mice (n = 8) were used to determine the iron status at the systemic and tissue levels and lipid metabolism and inflammatory signaling in adipose tissue. The same mice were used to evaluate cold tolerance (4°C) for 3 h. For assessing adaptive thermogenesis, mice (n = 5) received an intraperitoneal injection of β3-adrenoceptor agonist CL316243 (CL) for 5 d. ResultsCompared with the IA group, the ID group had nonanemic iron deficiency, lower serum ferritin (42.8%, P < 0.01), and greater weight gain (8.67%, P < 0.05) and insulin resistance (159%, P < 0.01), partly due to reduced AMP-activated protein kinase activation (61.0%, P < 0.05). Upon cold exposure, the ID group maintained a core body temperature 2°C lower than the IA group. The ID group had lower iron content (47.0%, P < 0.01) in the inguinal adipose tissue (iWAT) than the IA group, which was associated with impaired adaptive thermogenesis. In response to CL, ID mice showed decreased heat production (P < 0.01) and defective upregulation of beige adipocyte-specific markers, including uncoupling protein 1 (41.1%, P < 0.001), transferrin receptor 1 (47.5%, P < 0.001), and mitochondrial respiratory chain complexes (P < 0.05) compared with IA mice. ConclusionsDietary iron deficiency deregulates iron balance in the iWAT and impairs adaptive thermogenesis, thereby escalating the diet-induced weight gain in C57BL/6 mice.

Diet-Induced Non-anemic Iron Deficiency Attenuates Adaptive Thermogenesis via Defective Iron Metabolism of Adipose Tissue in C57BL/6 Mice

ObjectivesIron deficiency is one of the most common nutrient deficiencies worldwide. Adaptive thermogenesis plays a critical role for maintaining energy homeostasis. Currently, the role of dietary iron deficiency on adaptive thermogenesis is unknown. We hypothesized that dietary iron deficiency attenuates adaptive thermogenesis via dysfunctional iron metabolism in adipose tissue. MethodsC57BL/6 male mice were fed a diet containing either adequate-iron (Cont: 35ppm) or deficient-iron (ID: 3ppm) throughout for 14 weeks and the last 10 weeks were fed a high-fat diet to induce obesity. Systemic iron status was evaluated by measuring serum ferritin, hemoglobin (Hb), and hematocrit (HCt). The iron content of adipose tissue was determined by ICP-MS spectrometer. The protein and gene expressions related to iron-handling and thermogenesis were measured in adipose tissue by Western blot analysis and qPCR. To assess adaptive thermogenic function, mice were exposed to cold temperature acutely (3 hour) or administrated with β3-adrenoceptor agonist (CL) for 5 days. The core body temperature and thermogenesis were determined by a rectal thermometer and infrared camera, respectively. ResultsThe ID mice displayed a non-anemic iron deficiency with reduced serum ferritin (p < 0.01), increased weight gain (p < 0.05) and decreased insulin sensitivity (p < 0.05) compared to Cont. The iron content was significantly reduced in the inguinal (iWAT, p < 0.05) of the ID mice, which was linked with reduced thermogenic heat release upon acute cold treatment. In addition, ID mice markedly reduced the adipose tissue browning and thermogenic heat release upon CL stimulation compared with Cont. In terms of iron metabolism, CL-induced coordinated iron uptake for mitochondrial biogenesis in the iWAT was impaired in the ID mice, which was comparable to inflammation-mediated defective adipose tissue browning and thermogenesis. ConclusionsIron deficiency induced visceral obesity and compromised thermogenic function due to defective iron metabolism in C57BL6 mice. Funding SourcesNational Institutes of Health Grant 1R21HD094273.

1276. Evaluation of CD377, a Novel Antiviral Fc-Conjugate (AVC), In Vitro Activity and In Vivo Efficacy in Immune-Competent and -Deficient (SCID) Lethal Mouse Models

BackgroundCidara’s AVCs are novel, potent, antiviral agents conjugated with the Fc domain of human IgG1. CD377 is an AVC development candidate being evaluated for prevention and treatment of seasonal and pandemic influenza, including in immune-deficient populations unable to benefit from vaccination. We evaluated CD377 in vitro and in SCID mice to determine the impact of compromised immune status on efficacy.MethodsCD377 and comparators (oseltamivir [OS], zanamivir [ZA], baloxavir marboxil [BM]) were evaluated in vitro by neuraminidase inhibition (NAI), and cytopathic effect (CPE) assays. The pharmacokinetics (PK) and efficacy of CD377 were determined in immune-competent (IC; BALB/c) and immune-deficient (ID; BALB/c SCID) mice. Efficacy was assessed by intranasal challenge at 3x the LD95 of influenza A/Puerto Rico/8/1934 (H1N1), followed by a single subcutaneous (SC) dose of CD377, 2 hours post-challenge. The SCID study also evaluated the efficacy of BM at 3 mg/kg (BID x 1 day). Body weights (BW) were monitored for 21 days, with 20% BW loss recorded as mortality.ResultsIn vitro evaluation by NAI showed CD377, OS, and ZA to be approximately equipotent, with IC50 values between 0.5 and 1.7 nM. However, by CPE, CD377 was ~4.5-fold more active than BM and >1,000-fold more active than OS and ZA.In vivo, the PK of CD377 was found to be comparable in IC and ID mice. In subsequent efficacy studies, CD377 was protective at 0.1 mg/kg in IC mice (P=0.0020 vs. vehicle), while control groups fully succumbed to infection by Day 7 (Fig. 1A).In a similar study with ID mice, CD377 dosed at 0.1 mg/kg was also fully protective (P=0.0020). In contrast, mice treated with 6 mg/kg (total dose) of BM were only partially protected until day 13 (40% mortality by study end) (Fig. 1B). The potency of CD377 was further supported by BW data, which mirrored the survival data in both studies.ConclusionCD377 exhibited potent in vitro activity and had similar PK in IC and ID mice. In efficacy studies, CD377 demonstrated robust potency in both IC and ID mouse models at equivalent doses (0.1 mg/kg, SC, single dose). These results support further development of CD377 as a novel antiviral for the prevention and treatment of influenza infection, including in people with immune deficiencies and higher risk of infection.Balb/c and Balb/c SCID DisclosuresJames Levin, PhD, Cidara Therapeutics (Shareholder) Karin Amundson, B.S., Cidara Therapeutics (Shareholder) Allen Borchardt, PhD, Cidara Therapeutics (Employee) Thanh Lam, PhD, Cidara Therapeutics (Shareholder) Tom Brady, PhD, Cidara Therapeutics (Shareholder) Elizabeth Abelovski, B.S., Cidara Therapeutics (Shareholder) Simon Döhrmann, PhD, Cidara Therapeutics (Shareholder) Jason Cole, PhD, Cidara Therapeutics (Shareholder) Voon Ong, PhD, Cidara Therapeutics, Inc. (Employee, Shareholder) Les Tari, PhD, Cidara Therapeutics (Shareholder)

Iron deficiency without anemia is responsible for decreased left ventricular function and reduced mitochondrial complex I activity in a mouse model

BackgroundIron deficiency (ID), with or without anemia, is frequent in heart failure patients, and iron supplementation improves patient condition. However, the link between ID (independently of anemia) and cardiac function is poorly understood, but could be explained by an impaired mitochondrial metabolism. Our aim was to explore this hypothesis in a mouse model. Methods and resultsWe developed a mouse model of ID without anemia, using a blood withdrawal followed by 3-weeks low iron diet. ID was confirmed by low spleen, liver and heart iron contents and the repression of HAMP gene coding for hepcidin. ID was corrected by a single ferric carboxymaltose (FCM) injection (ID + FCM mice). Hemoglobin levels were similar in ID, ID + FCM and control mice. ID mice had impaired physical performances and left ventricular function (echocardiography). Mitochondrial complex I activity of cardiomyocytes was significantly decreased in ID mice, but not complexes II, III and IV activities. ID + FCM mice had improved physical performance, cardiac function and complex I activity compared to ID mice. Using BN-PAGE, we did not observe complex I disassembly, but a reduced quantity of the whole enzyme complex I in ID mice, that was restored in ID + FCM mice. ConclusionsID, independently of anemia, is responsible for a decreased left ventricular function, through a reduction in mitochondrial complex I activity, probably secondary to a decrease in complex I quantity. These abnormalities are reversed after iron treatment, and may explain, at least in part, the benefit of iron supplementation in heart failure patients with ID.

Alterations in B cell development, CDR-H3 repertoire and dsDNA-binding antibody production among C57BL/6 ΔD−iD mice congenic for the lupus susceptibility loci sle1, sle2 or sle3

Systemic lupus erythematosus (SLE) is an autoimmune disease that reflects a failure to block the production of self-reactive antibodies, especially those that bind double-stranded DNA (dsDNA). Backcrossing the lupus-prone NZM2410 genome onto C57BL/6 led to the identification of three genomic intervals, termed sle1, sle2 and sle3, which are associated with lupus susceptibility. We previously generated a C57BL/6 strain congenic for an immunoglobulin DH locus (ΔD–iD) that enriches for arginine at dsDNA-binding positions. We individually introduced the ΔD–iD allele into the three sle strains to test whether one or more of these susceptibility loci could affect the developmental fate of B cells bearing arginine-enriched CDR-H3s, the CDR-H3 repertoire created by the DH and the prevalence of dsDNA-binding antibodies. We found that the combination of the ΔD–iD allele and the sle1 locus led to a decrease in mature, recirculating B cell numbers and an increase in marginal zone cell numbers while maintaining a highly charged CDR-H3 repertoire. ΔD–iD and sle2 had no effect on peripheral B cell numbers, but the CDR-H3 repertoire was partially normalized. ΔD–iD and sle3 led to an increase in marginal zone B cell numbers, with some normalization of hydrophobicity. Mice with ΔD–iD combined with either sle1 or sle3 had increased production of dsDNA-binding IgM and IgG by 12 months of age. These findings indicate that the peripheral CDR-H3 repertoire can be categorically manipulated by the effects of nonimmunoglobulin genes.

The Role Of Liver Endothelial Sinusoidal Cells In Iron-Mediated Bmp6 Regulation

IntroductionIron-dependent regulation of hepcidin responds to circulating (holo-Tf) and hepatic iron. Although the signals that modulate hepcidin according to holo-Tf are still unknown, hepatic iron regulates hepcidin expression through transcriptional modulation of Bmp6. Bmp6 is controlled by iron only in the liver. Heterogeneous cell types with different functions in the liver include hepatocytes (HC) and non-parenchymal cells as Kupffer cells (KC), sinusoidal endothelial cells (LSEC), stellate cells. It has been reported that LSEC and KC, but not HC, upregulate Bmp6 independently of intracellular iron (Enns et al., Plos One 2013). Here we extend this observation analyzing Bmp6 regulation in isolated liver cells after acute and chronic changes of the iron status in wild type mice. In addition we studied pathogenic models of iron deregulation due to low (iron loaded Hjv KO mice) and high (iron deficient Tmprss6 KO mice) hepcidin. MethodsMice were maintained in accordance with the European Union guidelines. The study was approved by the IACUC of San Raffaele Scientific Institute, Milan, Italy. For chronic changes of the iron status, four weeks-old male mice were maintained an iron-replete (IB, 200 mg/kg iron), iron-deficient diet (ID, <3 mg /kg iron) or iron-loaded (IL, 8.3 g/kg iron) diet for 3 weeks. To induce acute iron changes, we used two approaches: iron dextran injection (1 g/kg body weight i.p.) or 2 weeks ID diet followed by 1, 3 and 6 days of IB, ID or IL diet. Hjv and Tmprss6 KO mice were studied in basal conditions at 7-8 weeks. Liver cells were isolated according to Liu et al. (Proteomics 2011) and cell purity validated by mRNA expression of specific genes. RNA was extracted using RNeasy Mini kit. Total RNA was retro-transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem). Gene expression levels were measured by quantitative real-time PCR using TaqMan Gene Expression Master Mix (Applied Biosystem). Transferrin saturation (TS) and liver iron content (LIC) was measured as previously described (Pagani et al. Blood 2011). ResultsIn wild type animals hepcidin in basal condition is expressed exclusively in HC, whereas Bmp6 is expressed in non-parenchymal cells, mainly LSEC. In all cell types, Bmp6 levels inversely correlate with Tfr1 expression, suggesting that cellular iron influences Bmp6. In mice chronically maintained an IB, ID and IL diet HC, LSEC and KC modulate Bmp6 according to both cellular iron and TS. To investigate which cell type first regulates Bmp6, we injected wild type mice with a single dose of iron dextran and separated cells 3 and 6 hrs post injection. At 6 hrs, when TS and LIC are increased and Hamp is activated in HC, only LSEC upregulate Bmp6. As for the pathogenic models, Bmp6 is upregulated in HC, LSEC and KC cells in Hjv KO mice and downregulated in Tmprss6 KO animals. However, at difference with HC, regulation of Bmp6 in LSEC and KC is independent on cellular iron (as shown by Tfr1 mRNA), and related to TS in both animal models. To distinguish between the contribution of cellular and circulating iron in the regulation of Bmp6, we fed mice previously treated with an ID diet, with an ID, IB and IL diet for 1-3-6 days. At day 1, TS increases but LIC does not change in IB and ID mice while total liver Bmp6 is modulated according to TS. These results will be analyzed in separated cells. In vitro, exogenous BMP6 transcriptionally activates BMP6 expression in hepatoma-derived cells, even at short time point, suggesting that a paracrine mechanism contributes to BMP6 regulation. ConclusionsIn our hands Bmp6 expression is modulated not only in LSEC and KC but also in HC in animals fed a variable iron content diet. LSEC are the first iron “sensor” that, through Bmp6, activates hepcidin expression in HC. Bmp6 in LSEC and KC responds mainly to TS changes. We also suggest that an autocrine/paracrine mechanism activates liver Bmp6 to amplify signaling for hepcidin increase. Disclosures:No relevant conflicts of interest to declare.

MRET Water Effect in the TgCRND8 Transgenic Amyloid Mice Models

Th is particular art icle relates to study in vivo regarding the effect of M RET Activated water in Transgenic Amylo id Mice models. It provides some evidence on how MRET Activated water with the mod ified molecu lar structure, physical and electrodynamic characteristics may enhance specific mo lecular mechanisms in living cells. The anomalous proton activity, electrodynamic characteristics and viscosity of MRET Activated water provide some evidence rega rding its possible effect on electrical activity and proper function of the cells. Most cells and tissues have electrical p roperties relevant to their na tural function. The living cells and t issues have rather co mplex structure, consisting of the fold ing memb ranes, the specialized con- nections, and organelles. The localization of electrical properties is particu larly important, since each of the co mplex stru ctures must be expected to have a specific ro le in the electrical function of the tissue. MRET Activ ated water with modified proton activity and electrical conductivity can also have ability to enhance a proton pump activity of the cells. As a result it may lead to the restoration of the transduction signaling and the restoration of normal cellu lar funct ions. The clinical study in vivo in Transgenic Amyloid M ice models conducted at Toronto University proves the validity of the proposed hypothesis.

The effects of dietary iron deprivation on murine circadian sleep architecture

ObjectivesIron deficiency is considered a putative cause for restless legs syndrome (RLS), a human sensorimotor disorder characterized by a circadian presentation of symptoms during the evening hours that disrupts one’s ability to sleep. We sought to evaluate the sleep–wake effects of diet-induced iron deficiency in mice as an animal model of RLS. To this end, we hypothesized that the iron-deprived mice would exhibit a sleep–wake circadian pattern characteristic of the human syndrome: increased wakefulness during the hours immediately preceding the sleep-predominant period. MethodsFollowing weaning at post-natal day (PND) 21, C57BL/6J mice were assigned to one of two dietary treatments: iron-deficient (ID, n=7) or iron-adequate (i.e., control, CTL, n=6). At PND 44, the mice were surgically instrumented for polysomnographic (PSG) recording, and data were collected at young adulthood: PNDs 59 and 60. Sleep–wake architecture was characterized for the 12-h light and dark periods and also for six consecutive 4-h blocks comprising a 24-h day. ResultsThe ID mice showed marked increases in wake time in the 4-h period prior to lights-on; both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep were reduced. In contrast, sleep–wake activity did not differ across the 12-h light period. ConclusionsDietary iron deficiency in mice elicited increases in wakefulness during a particular circadian time point that corresponds to the period during which RLS symptoms would maximally disturb sleep onset and progression in humans. These data indicate that iron-deficient mice may provide a potentially useful animal model for RLS.

Hyperglycemia, impaired glucose tolerance and elevated glycated hemoglobin levels in a long-lived mouse stock

We have previously demonstrated that two wild-derived stocks of mice, Idaho and Majuro, are significantly longer-lived than mice of a control stock (DC) generated as a four-way cross of commonly used laboratory strains of mice. This study provides independent confirmation of this earlier finding, as well as examining serum glucose, insulin, leptin, glycated hemoglobin (GHb), cataract severity, and glucose tolerance levels in each of the stocks. Both the mean (+20%) and maximum (+13%) life span of the Idaho mice were significantly increased relative to the DC stock, while in the Majuro mice only maximum (+15%) life span was significantly increased. In addition, Majuro mice were hyperglycemic in both the fed and fasted states compared both to laboratory-derived and Idaho stocks, had significantly elevated GHb levels and cataract scores, and were glucose intolerant although serum insulin levels did not differ between stocks. Body weight and body mass index (BMI)-corrected leptin levels were also dramatically (1.5–3-fold) higher in the Majuro mice. The longevity of Id mice was not accompanied by changes in serum glucose and insulin levels, or glucose tolerance compared to DC controls, although GHb levels were significantly lower in the Idaho mice. Taken together, these findings suggest that neither a reduction of blood glucose levels nor an increase in glucose tolerance is necessary for life span extension in mice.

Iron deficiency and in vitro iron chelation reduce the expression of cluster of differentiation molecule (CD)28 but not CD3 receptors on murine thymocytes and spleen cells.

Cluster of differentiation molecule (CD)3 and CD28 receptors play crucial roles in T-lymphocyte proliferation. Fe deficiency in man and animals impairs T-lymphocyte proliferation by unknown mechanisms. To test the hypothesis that reduced CD3 and CD28 expression is one of them, thymocytes and splenocytes from control (C; n 24), Fe-deficient (ID; n 24), pair-fed (PF; n 24), and ID mice that were Fe-repleted for 3 (R3; n 24) or 14 d (R14; n 12) were labelled with anti-CD3-fluorescein isothiocyanate and anti-CD28-phycoerythrin antibodies. Positive cells were analysed by flow cytometry. Significant differences were observed among groups in the mean levels of haemoglobin and liver Fe stores (C=PF=R14>R3>ID; P<0.005). While Fe deficiency slightly increased the percentage of CD3+ splenocytes, it reduced that of CD28+ thymocytes in mice with thymus atrophy and splenomegaly (P<0.05). These changes were corrected by Fe repletion. CD28 mean fluorescence intensity (FI) was lower and CD3 FI was higher in lymphocytes from R3 and ID, especially those with splenomegaly, than in those from R14 and PF mice (P<0.05). In vitro Fe chelation by deferoxamine (60 min) significantly decreased CD28 expression (P<0.05), and slightly increased that of CD3 (P>0.05). Spleen cell proliferative responses to concanavalin A and anti-CD3+/-anti-CD28 were reduced by Fe deficiency (ID</=R3<C=PF<R14; P<0.05); and they correlated with FI and percentages of CD3+ and CD28+ cells (r< or =0.69; P<0.05). Indicators of Fe status negatively correlated with CD3 FI (r-0.23), but positively correlated with CD28 FI (r< or =0.44; P<0.05). Data suggest that altered CD28 expression may contribute to reduced T-cell proliferation during Fe deficiency.

Longer life spans and delayed maturation in wild-derived mice.

Nearly all the experimental mice used in aging research are derived from lineages that have been selected for many generations for adaptation to laboratory breeding conditions and are subsequently inbred. To see if inbreeding and laboratory adaptation might have altered the frequencies of genes that influence life span, we have developed three lines of mice (Idaho [Id], Pohnpei [Po], and Majuro [Ma]) from wild-trapped progenitors, and have compared them with a genetically heterogeneous mouse stock (DC) representative of the laboratory-adapted gene pool. Mean life span of the Id stock exceeded that of the DC stock by 24% (P < 0.00002), and maximal life span, estimated as mean longevity of the longest-lived 10% of the mice, was also increased by 16% (P < 0.003). Mice of the Ma stock also had a significantly longer maximal longevity than DC mice (9%, P = 0.04). The longest-lived Id mouse died at the age of 1450 days, which appears to exceed the previous longevity record for fully fed, non-mutant mice. The life table of the Po mice resembled that of the DC controls. Ma and Id mice differ from DC mice in several respects: both are shorter and lighter, and females of both stocks, particularly Id, are much slower to reach sexual maturity. As young adults, Id mice have lower levels of insulin-like growth factor 1 (IGF-I), leptin, and glycosylated hemoglobin compared with DC controls, implicating several biochemical pathways as potential longevity mediators. The results support the idea that inadvertent selection for rapid maturation and large body size during the adaptation of the common stocks of laboratory mice may have forced the loss of natural alleles that retard the aging process. Genes present in the Id and Ma stocks may be valuable tools for the analysis of the physiology and biochemistry of aging in mice.

Resting small B cells present endogenous immunoglobulin variable-region determinants to idiotope-specific CD4(+) T cells in vivo.

Antigenic determinants localized within the highly diversified V-regions of Ig are called idiotopes (Id). Processed Id-peptides can be presented on MHC class II molecules to CD4(+) T cells. If B cells present their endogenous Id-peptides, T cell activation could occur in the absence of nominal antigen, a potentially important process in T-B cooperation and immune regulation. To test this idea, we used mice made transgenic for a lambda2 L-chain (Id(+) mice). Another transgenic mouse strain expresses TCR transgenes with specificity for the Id (lambda2), presented on MHC class II molecules. When highly purified sorted Id(+) B cells and Id-specific T cells were sequentially injected into MHC syngeneic SCID host, T cell became blastoid, CD69(+) and proliferated. To exclude any role of host APC, MHC incompatible Rag2(- / -) mice (H-2(b)) were used as recipients for the Id(+) B and Id-specific T cells, with similar results. Exposure to extracellular Id(+) immunoglobulin (Ig) was not sufficient for Id priming of B cells in vivo, highlighting the preferential presentation of Id peptides derived from endogenous Ig, by B cells. The results suggest that B cells presenting Id self-peptides generated by V(D)J recombinations or somatic mutations may directly stimulate T cell in vivo in the absence of conventional antigen.

Impaired protein kinase C activation as one of the possible mechanisms of reduced lymphocyte proliferation in iron deficiency in mice

We investigated the effects of iron deficiency in mice on protein kinase C (PKC) activation, an enzyme required for optimal lymphocyte proliferation. C57BL/6 mice were fed either an iron-deficient diet (ID; 10 mg Fe/kg diet), a control diet (C; 50 mg/kg diet), or were pair fed (PF) to ID mice for 34 d. PKC activity was studied in spleen cells by histone phosphorylation. Iron deficiency significantly reduced cytosol activity in unstimulated cells and membrane-bound activity in cells stimulated by concanavalin A (Con A) or phorbol-12-myristate-13-acetate (PMA), and the ratio of membrane-bound over cytosol activity in mitogen-stimulated cells. In PF mice the ratio of membrane-bound activity to cytosol activity was greater than normal in Con A-treated cells and only slightly decreased in PMA-treated cells. PKC activity positively correlated with iron status. We conclude that reduced PKC activity and poor translocation results in aberrant signal transduction, which in turn might be responsible for the impaired lymphocyte proliferation associated with iron deficiency.

Mitogenic response of lymph nodes and spleen lymphocytes from mice with moderate and severe iron deficiency anemia

To define the effects of moderate and severe iron deficiency (Hb: 4–10.4 g/dl) on mitogenic response of lymph nodes (LN) and splenic (S) lymphocytes, C57BL/6 weanling female mice were fed an iron deficient (<5ppm; ID), an iron adequate diet (30 cpm, C) or restricted amounts of the adequate diet (pairfed:PF) for 45–56 days. 3H-thymidine incorporation into DNA was measured in the presence of Con A, PHA and LPS. In both lymphoid organs and with three mitogens tested, iron deficiency significantly reduced blastogenic response (stimulation indices=SI) by 23% to 93%. However, splenic lymphocytes were more affected than lymph nodes. In the spleen, SI positively correlated with Hb and liver iron stores but negatively with spleen weight. Total cpm in response to Con A and LPS were reduced in both S and LN; but those in response to PHA were reduced only in moderately deficient mice. In S but not LN, underfeeding in the overall population of PF mice significantly reduced SI in response to Con A but not PHA or LPS. In ID mice, thymus weight correlated with body weight, (r=0.88), Hb levels (r=0.50) and liver iron stores (r=0.34). While iron deficiency significantly decreased IgG and IgM levels, underfeeding in PF significantly reduced IgA levels and only slightly IgM levels. We conclude that even moderate iron deficiency anemia (11 g/dl<Hb<8 g/dl) impairs mitogenic response in both S and LN.

Segregation at a locus determining an immunoglobulin genetic marker for the light chain variable region affects inheritance of expression of an idiotype

Previous investigations have demonstrated close genetic linkage between loci governing expression of strain-specific idiotypes and immunoglobulin heavy (H) chain allotype (i.e., the C(H) locus). This linkage is presumed to reflect polymorphism of V(H) genes (or of their expression) linked to the polymorphic C(H) locus. That there was no apparent involvement of light (L) chain loci (thought to be unlinked to H chain) in inheritance of the idiotype-positive (Id(+)) phenotype was surprising, because the L chain is required for formation of each of the idiotypes studied at the chemical level. However, previous studies involving backcrosses of F(1)(Id(+) x Id(-)) mice to the Id(-) parental strain have never employed as the Id(-) parent one of several inbred strains shown by G. M. Edelman and P. D. Gottlieb [(1970) Proc. Natl. Acad. Sci. USA 67, 1191-1199] to express a V(L)-region polymorphism. Among backcrosses performed in the present study, one involved the A/J strain as the Id(+) parent and the PL/J strain, one of the several strains with and L chain polymorphism, as the Id(-) parent. Whereas in three other backcrosses performed, idiotype expression segregated with H chain allotype, in the backcross to PL/J, all mice producing the characteristic A/J Id(+) phenotype were A/J allotype(+), but not all A/J allotype(+) mice were Id(+). Typing of backcross progeny for the Ly-3 thymocyte alloantigens, controlled by a locus closely linked to that governing the L chain polymorphism (called the VK-1 locus), indicated that only the Ly-3 heterozygotes expressed the characteristic A/J Id(+) phenotype. Thus, all Id(+) backcross mice inherited both the H chain allotype and the Ly-3 locus (and closely linked L chain-related locus) from the Id(+) A/J strain. This suggests that, when strains with L chain polymorphisms are included in genetic studies of idiotype expression, segregation of loci governing L chain expression may be found to contribute to inheritance of the Id(+) character. In addition, these studies may be taken as further evidence that the V(L)-region repertoire of the PL/J strain may differ considerably from that of most other inbred strains of mice.