ABSTRACT
STUDY QUESTION: Does the administration of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine have an association with ovarian reserve as expressed by circulating anti-Müllerian hormone (AMH) levels? SUMMARY ANSWER: Ovarian reserve as assessed by serum AMH levels is not altered at 3 months following mRNA SARS-CoV-2 vaccination. WHAT IS KNOWN ALREADY: A possible impact of SARS-CoV-2 infection or vaccination through an interaction between the oocyte and the somatic cells could not be ruled out, however, data are limited. STUDY DESIGN, SIZE, DURATION: This is a prospective study conducted at a university affiliated tertiary medical center between February and March 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Study population included reproductive aged women (18-42 years) that were vaccinated by two Pfizer-BioNTech Covid-19 vaccines (21 days apart). Women with ovarian failure, under fertility treatments, during pregnancy, previous Covid-19 infection or vaccinated were excluded from the study. Blood samples were collected for AMH levels before the first mRNA vaccine administration. Additional blood samples after 3 months were collected for AMH and anti-Covid-19 antibody levels. Primary outcome was defined as the absolute and percentage change in AMH levels. MAIN RESULTS AND THE ROLE OF CHANCE: The study group consisted of 129 women who received two mRNA vaccinations. Mean AMH levels were 5.3 (±SD 4.29) µg/l and 5.3 (±SD 4.50) µg/l at baseline and after 3 months, respectively (P = 0.11). To account for possible age-specific changes of AMH, sub-analyses were performed for three age groups: <30, 30-35 and >35 years. AMH levels were significantly lower for women older than 35 years at all times (P = 0.001 for pre and post vaccination AMH levels versus younger women). However, no significant differences for the changes in AMH levels before and after vaccinations (Delta AMH) were observed for the three age groups (P = 0.46). Additionally, after controlling for age, no association was found between the degree of immunity response and AMH levels. LIMITATIONS, REASONS FOR CAUTION: Although it was prospectively designed, for ethical reasons we could not assign a priori a randomized unvaccinated control group. This study examined plasma AMH levels at 3 months after the first vaccination. It could be argued that possible deleterious ovarian and AMH changes caused by the SARS-CoV-2 mRNA vaccinations might take effect only at a later time. Only longer-term studies will be able to examine this issue. WIDER IMPLICATIONS OF THE FINDINGS: The results of the study provide reassurance for women hesitant to complete vaccination against Covid 19 due to concerns regarding its effect on future fertility. This information could be of significant value to physicians and patients alike. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by Sheba Medical Center institutional sources. All authors have nothing to disclose. TRIAL REGISTRATION NUMBER: The study protocol was approved by the 'Sheba Medical Center' Ethical Committee Review Board (ID 8121-21-SMC) on 8 February 2021 and was registered at the National Institutes of Health (NCT04748172).
Subject(s)
COVID-19 Vaccines , COVID-19 , Adult , Anti-Mullerian Hormone , COVID-19/prevention & control , COVID-19 Vaccines/adverse effects , Female , Humans , Pregnancy , Prospective Studies , SARS-CoV-2 , Vaccines, Synthetic , mRNA VaccinesABSTRACT
Short structural variants-variants other than single nucleotide polymorphisms-are hypothesized to contribute to many complex diseases, possibly by modulating gene expression. However, the molecular mechanisms by which noncoding short structural variants exert their effects on gene regulation have not been discovered. Here, we study simple sequence repeats (SSRs), a common class of short structural variants. Previously, we showed that repetitive sequences can directly influence the binding of transcription factors to their proximate recognition sites, a mechanism we termed non-consensus binding. In this study, we focus on the SSR termed Rep1, which was associated with Parkinson's disease (PD) and has been implicated in the cis-regulation of the PD-risk SNCA gene. We show that Rep1 acts via the non-consensus binding mechanism to affect the binding of transcription factors from the GATA and ELK families to their specific sites located right next to the Rep1 repeat. Next, we performed an expression analysis to further our understanding regarding the GATA and ELK family members that are potentially relevant for SNCA transcriptional regulation in health and disease. Our analysis indicates a potential role for GATA2, consistent with previous reports. Our study proposes non-consensus transcription factor binding as a potential mechanism through which noncoding repeat variants could exert their pathogenic effects by regulating gene expression.
Subject(s)
Microsatellite Repeats/physiology , Parkinson Disease/genetics , Polymorphism, Single Nucleotide , alpha-Synuclein/genetics , Binding Sites/genetics , GATA Transcription Factors/metabolism , GATA2 Transcription Factor/metabolism , Gene Expression Regulation , Genetic Predisposition to Disease , Humans , Microsatellite Repeats/genetics , Parkinson Disease/pathology , Sequence Analysis, DNA , ets-Domain Protein Elk-1/metabolismABSTRACT
In order to examine sex-specific differences in the association of body mass index (BMI) and hypertension, we conducted a retrospective, cross-sectional study of 717 812 (402 914 men and 314 898 women) Israeli Jewish adolescents aged 16.0-19.99 years medically screened for military service. A diagnosis of hypertension was established per history or if a mean of 10 separate blood pressure measurements exceeded 140/90, following an initial measurement higher than 140/90. Weight and height were measured. Prevalence of hypertension was 0.42% in men and 0.05% in women. In men, BMI was significantly associated with hypertension from the third decile (odds ratio [OR] 1.67, 1.06-2.65) up to the 10th decile (OR 30.17, 20.83-43.69). In women, we observed a significantly increased risk for hypertension in the ninth decile (OR 3.82, 1.42-10.22) and in the 10th decile (OR 18.92, 7.7-46.51), with no visible trend in lower deciles. BMI effects on hypertension prevalence are different in male and female adolescents.
Subject(s)
Body Mass Index , Hypertension/epidemiology , Sex Factors , Adolescent , Blood Pressure/physiology , Body Weight , Cross-Sectional Studies , Female , Humans , Israel , Male , Prevalence , Retrospective Studies , Risk Factors , Young AdultABSTRACT
Scarce data are available on epidemiology of varicocoele, the most common surgically correctable cause of male infertility. The objectives of this study were to evaluate the association between body mass index (BMI) and varicocoele and to assess trends in prevalence over time. We conducted a nationwide population-based long-term (1967-2010) study among 1 323 061 Israeli adolescent males using data from mandatory medical examination. BMI was grouped into underweight, normal weight, overweight and obese categories by percentiles adjusted for age in months and by further classification to five categories within normal weight. Univariable and multivariable logistic regression models were constructed, adjusting for possible confounders. Varicocoele prevalence (N = 47 398) increased during the study period from 1.6% for the 1950-1954 birth cohort to 4.6% for the 1990-1993 birth cohort, with the steepest rise in the normal weight group. Varicocoele unadjusted rates were highest (4.1%) among underweight and lowest (1.6%) among obese. In a multivariable model, adjusted for birth cohort, height, age and socio-demographic factors, we found a decreased risk for varicocoele in the overweight group [odds ratio (OR) = 0.51, 95% confidence interval (CI): 0.49, 0.54] and the obese group (OR = 0.34, 95% CI: 0.32, 0.37), compared with the normal weight group. Within the normal weight group, a monotonic inverse association between BMI percentile and varicocoele was observed, most notable among 75-84.9 percentile compared to 25-49.9 percentile (OR = 0.65, 95% CI: 0.63, 0.68). In conclusion, varicocoele is common among adolescents in Israel, and its prevalence had increased in recent decades, providing clues to direct further andrological research on the role of modern lifestyle and environment in the aetiology of varicocoele. BMI, across percentiles, was found to be monotonically inversely associated with varicocoele, thus directing research and clinical efforts.
Subject(s)
Body Mass Index , Obesity/epidemiology , Varicocele/epidemiology , Adolescent , Humans , Infertility, Male , Israel/epidemiology , Male , Oligospermia , Prevalence , Semen Analysis , Spermatozoa/abnormalities , Varicocele/surgery , Young AdultABSTRACT
BACKGROUND: The epidemic of obesity has been identified as a major source of morbidity, not just in developed countries but globally, in adults as well as at younger ages. OBJECTIVE: The aims of this study were to describe trends in obesity and overweight in Israeli adolescents and observe temporal changes and association by risk factors. METHODS: The research analyzed records of 2,148,342 Jewish adolescents, over a span of 44 years and included data for individual body measurements, place of residence, area of origin and education levels. Body mass index (BMI) was measured by professionals, calculated and categorized as overweight or obesity according to age- and gender-specific BMI curves established in recent years. We processed the data in multinomial logistic regression model and calculated odds ratios for various risk factors. RESULTS: Obesity and overweight are on the rise for male and female adolescents born from the mid-1960s onwards, and especially for men from the 1980s onwards. Risk factors for male adolescents include lower socioeconomic status, inferior education levels and Western origins (vs. Asian, African or Israeli origins). Risk modifiers for women were similar, except for African origins, which were associated with increased risk rather than decreased risk. Asian and Israeli origins were protective for both genders, and education was more strongly associated with obesity for women. CONCLUSIONS: We recommend stronger preventive efforts directed at adolescents as a whole, and particularly vulnerable groups with lower education levels and poverty, or those with specific geographical origins. Gender disparities are evident and should be considered in these efforts and in further research.
Subject(s)
Jews/statistics & numerical data , Obesity/epidemiology , Population Surveillance , Adolescent , Body Mass Index , Educational Status , Female , Humans , Israel/epidemiology , Male , Obesity/prevention & control , Odds Ratio , Risk Factors , Sex Factors , Social ClassABSTRACT
BACKGROUND: Measurements at the end of puberty of neonates short for gestational age (SGA-L) are scant. OBJECTIVE: To determine the correlation between birth length and weight in neonates, with height and weight at age 17 years. SUBJECTS AND METHODS: 385 full-term neonates, measuring less than 48 cm (SGA-L) and 585 full-term neonates, measuring 48 cm or greater (adequate birth length for gestational age; AGA-L) were included. 234 SGA-L and 359 AGA-L were identified at age 17 years. RESULTS: Comparison of the two groups revealed that both sexes born SGA-L were also shorter at age 17 years than those born AGA-L (girls 158.9 cm (SD 7.6) vs 164.2 cm (SD 64) (p<0.001) and boys 167.3 cm (SD 8.7) vs 173.8 cm (SD 7.1) (p<0.001)). The subjects born SGA-L also weighed significantly less than those born AGA-L (p<0.001) both at birth and at age 17 years. CONCLUSIONS: Children born SGA-L become short adults and weigh less at age 17 years than children with a normal birth length.
Subject(s)
Adolescent Development/physiology , Child Development/physiology , Infant, Small for Gestational Age/growth & development , Adolescent , Aging/physiology , Anthropometry/methods , Birth Weight/physiology , Body Height/physiology , Body Weight/physiology , Female , Humans , Infant, Newborn , Male , Retrospective StudiesABSTRACT
BACKGROUND: 15-Lipoxygenase (15-LO) is a nonheme iron-containing enzyme that catalyzes the peroxidation of fatty acids. Herein, we studied the effect of 15-LO overexpression in the vascular endothelium on thymocyte apoptosis by evaluating thymuses from low-density lipoprotein receptor-deficient (LDL-RD) mice and LDL-RD/15-LO mice. Thymuses were evaluated by immunohistochemistry and by TUNEL whereas in vitro studies were carried out by employing freshly isolated thymocytes from the respective mice and evaluation of apoptosis by propidium iodide and annexin V cytometry. METHODS AND RESULTS: The apoptotic index in LDL-RD/15-LO mice was significantly higher than in the LDL-RD mice. In the thymic medulla the difference was smaller, although still significant. Freshly isolated thymus cells from LDL-RD/15-LO mice exhibited a higher rate of spontaneous cell death than controls. Incubation of thymus cells in the presence of the cell-permeable caspase-3 inhibitor DEVD-CMK resulted in a decrease in the frequency of apoptotic cells in LDL-RD/15-LO thymocytes, whereas no effect was evident in control thymocytes. The antioxidant N-acetylcysteine causes the increase in apoptosis in both groups. CONCLUSION: LDL-RD/15-LO mice exhibit increased thymocyte apoptosis both in vivo and in vitro. These findings may suggest a role for 15-LO in the natural selection of thymocytes.
Subject(s)
Arachidonate 15-Lipoxygenase/physiology , Clonal Deletion/physiology , Endothelium, Vascular/metabolism , T-Lymphocytes/cytology , Thymus Gland/cytology , Acetylcysteine/pharmacology , Animals , Antioxidants/pharmacology , Apoptosis , Arachidonate 15-Lipoxygenase/genetics , Caspase 3 , Caspase Inhibitors , Cells, Cultured/cytology , Cells, Cultured/drug effects , Cells, Cultured/enzymology , Cysteine Proteinase Inhibitors/pharmacology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Oligopeptides/pharmacology , Receptors, LDL/deficiency , Receptors, LDL/genetics , Thymus Gland/blood supplyABSTRACT
Kawasaki disease (KD) is a systemic vasculitis with cardiac and noncardiac complications. Anti--endothelial cell antibodies (AECA) are found among many patients with KD. The aim of this study was to investigate the pathogenic role of AECA in KD using in vitro and in vivo experimental models. F(ab)2 fragments of IgG-AECA and IgM-AECA were affinity purified from a patient with active KD. Their endothelial binding and ability to induce a pro-adhesive and a pro-inflammatory phenotype were evaluated in vitro. Twenty Balb/C mice were immunized with KD-AECA or with control Ig (N-Ig) to induce AECA in a murine model by the idiotypic manipulation method. Both KD-AECA isotypes bind significantly to human umbilical vein endothelial cell (HUVEC) compared to N-Ig. The in vitro activity was demonstrated by the antibodies ability to activate endothelial cells resulting in increased IL-6 secretion, adhesion molecule expression and monocytic cell line (U937) adherence to HUVEC. Five of the mice that received KD-AECA developed murine AECA after 3 months. None of the mice that received N-Ig produced AECA. The murine AECA increased monocyte adhesion to EC in vitro, similarly to the AECA used for immunization. Furthermore, all the mice that developed AECA had proteinuria and IgG deposition in the renal mesangium. No histological or immunofluorescence evidence of cardiac vasculitis could be detected. AECA might play a role in the emergence of some of KD manifestations.
Subject(s)
Autoantibodies/immunology , Endothelium, Vascular/immunology , Mucocutaneous Lymph Node Syndrome/immunology , Animals , Binding, Competitive , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cells, Cultured , Child, Preschool , Humans , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Kidney/immunology , Mice , Mice, Inbred BALB C , Monocytes/immunology , Mucocutaneous Lymph Node Syndrome/diagnosis , Mucocutaneous Lymph Node Syndrome/pathology , U937 CellsABSTRACT
BACKGROUND: Human 15-lipoxygenase (LO) and its murine analogue 12/15-LO are capable of directly oxidizing esterified fatty acids in lipoproteins and phospholipids. Because these oxidized products possess atherogenic properties, it was suggested that LOs may be involved in enhancing atherogenesis. Previous in vivo tests of the role of LOs in atherogenesis animal models, however, have yielded conflicting results. METHODS AND RESULTS: Aiming to study the role of the 12/15-LO in murine atherogenesis, we crossed LDL-receptor-deficient mice (LDL-R(-/-)) with 12/15-LO-knockout mice and evaluated plaque formation 3 to 18 weeks after initiation of a high-fat diet. Atherosclerotic lesions were considerably reduced in the LDL-R/12/15-LO-double-knockout mice compared with LDL-R(-/-) mice at 3, 9, 12, and 18 weeks, at the aortic root as well as throughout the aorta. The cellular composition of plaques from mice deficient in 12/15-LO did not differ with respect to macrophage and T-lymphocyte content compared with plaques from 12/15-LO littermates. CONCLUSIONS: 12/15-LO plays a dominant role in promoting atherogenesis in LDL-R(-/-) mice.
Subject(s)
Arachidonate 12-Lipoxygenase/genetics , Arachidonate 12-Lipoxygenase/physiology , Arachidonate 15-Lipoxygenase/genetics , Arachidonate 15-Lipoxygenase/physiology , Arteriosclerosis/etiology , Receptors, LDL/genetics , Animals , Aorta/pathology , Arteriosclerosis/blood , Arteriosclerosis/pathology , Cholesterol/blood , Diet, Atherogenic , Leukocyte Count , Macrophages , Mice , Mice, Knockout , T-Lymphocytes , Triglycerides/bloodABSTRACT
OBJECTIVES: This study was designed to determine the role of cellular and humoral immune responses to heat shock protein 65 (HSP65) in murine atherosclerosis. BACKGROUND: Inflammatory processes appear to influence the progression of atherosclerosis. Immunization with HSP65 was previously shown to induce arteriosclerosis in rabbits and to enhance fatty-streak formation in mice. However, it has not been demonstrated directly whether HSP65-reactive antibodies and lymphocytes are separately capable of influencing lesion formation. METHODS: Low density lipoprotein-receptor deficient (LDL-RD) mice were immunized with HSP65 or control bovine serum albumin (BSA). Lymph-node cells, splenocytes and immunoglobulin G (IgG) were obtained from the immunized mice and transferred separately to six groups of syngenic LDL-RD mice. RESULTS: Adoptive transfer of HSP65-reactive lymph node cells increased fatty-streak formation in comparison with mice treated with BSA-primed cells. Similarly, transfer of splenocytes reactive with HSP65 led to enhanced fatty-streak generation compared with mice injected with BSA-sensitized splenocytes. Repeated intraperitoneal administration of IgG from serum of HSP65-immunized mice (every 10 days) enhanced fatty-streak formation in mice in comparison with their anti-BSA-IgG injected littermates. CONCLUSIONS: Antibodies and lymphocytes reactive to HSP65 promote fatty-streak formation in mice, providing direct evidence for the proatherogenic properties of cellular and humoral immunity to HSP65.
Subject(s)
Arteriosclerosis/immunology , Bacterial Proteins/immunology , Chaperonins/immunology , Receptors, LDL/immunology , Animals , Antibody Formation , Chaperonin 60 , Enzyme-Linked Immunosorbent Assay , Female , Immunity, Cellular , Immunoglobulin G/immunology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , T-Lymphocytes/immunology , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
The application of brief periods of heat stress prior to induction of various forms of tissue injury (ischemia-reperfusion, myocardial infarction, endothelial denudation) has been shown to result in preconditioning and attenuation of subsequent damage. Atherosclerosis represents a state of heightened response to injury at the level of the vessel wall, involving endothelial cells, smooth muscle cells, and macrophages. In the current study, we studied the effects of whole body hyperthermia (WBH) on diet-induced atherosclerosis in a murine model. Low-density lipoprotein receptor deficient mice were either exposed to a 30-min WBH (n = 10) or nontreated (n = 7). Animals were given a high-fat ("Paigen"-type) diet to speed the progression of atherosclerosis immediately following WBH for 6 weeks. Aortic and plaque heat shock protein (HSP) 70, suggested to mediate thermotolerance, was assessed by immunohistochemisry and Western blot at different time points following induction of WBH. Aortic sinus plaque formation was significantly accelerated in WBH-treated mice (275,800 +/- 19,540 microm(2) ) in comparison with their control litters (152,100 +/- 18,200 microm(2); P = 0.0004). Plaque composition was also influenced by WBH as lesions were more mature and had an increased proportion of lipid core/fibrous cap accompanied by increased numbers of apoptotic cells. Total cholesterol and triglyceride levels were not affected significantly by WBH. HSP70 protein expression in the aortas was increased 30 min and 6 and 12 h following WBH induction. Thus, induction of WBH, which affords protection in models of arterial injury, appears to have a proatherogenic role in murine atherosclerosis, despite its upregulatory influence on the expression of HSP70.
Subject(s)
Arteriosclerosis/physiopathology , Fever/physiopathology , Heat Stress Disorders/physiopathology , Muscle, Smooth, Vascular/pathology , Receptors, LDL/physiology , Sinus of Valsalva/pathology , Animals , Apoptosis , Arteriosclerosis/genetics , Arteriosclerosis/pathology , Autoantibodies/blood , Diet, Atherogenic , Dietary Fats , Disease Progression , Fever/immunology , Fever/pathology , HSP70 Heat-Shock Proteins/immunology , Heat Stress Disorders/immunology , Heat Stress Disorders/pathology , Immunohistochemistry , In Situ Nick-End Labeling , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Receptors, LDL/deficiency , Receptors, LDL/geneticsABSTRACT
Gene therapy directed specifically to the vascular wall, particularly to angiogenic endothelial cells is a prerequisite in vascular disease treatment. Angiogenesis is a major feature in many pathological conditions including wound healing, solid tumors, developing metastases, ischemic heart diseases and diabetic retinopathy. In the present study we developed a tissue-specific gene therapy to the angiogenic blood vessels of tumor metastasis using an adeno-based vector containing the murine preproendothelin-1 (PPE-1) promoter. Genes activated by the PPE-1 promoter were highly expressed in bovine aortic endothelial cells in vitro. Systemic injection of the adenoviral vectors AdPPE-1-luciferase and AdCMV-luciferase to normal C57BL/6 mice, resulted in higher activity of PPE-1 promoter compared with CMV promoter in the aorta and vascularized tissues such as heart, kidney, lung and pancreas. Systemic administration of the adenoviral vector, in mice bearing Lewis lung carcinoma, resulted in high and specific activity of PPE-1 in the new vasculature of primary tumors and lung metastasis. Cellular distribution of the delivered gene revealed highest expression of GFP in angiogenic endothelial cells of the metastasis. We expect that this approach of 'vascular-directed' gene therapy will be applicable to both vascular diseases and cancer.
Subject(s)
Carcinoma, Lewis Lung/secondary , Endothelins/genetics , Endothelium, Vascular/metabolism , Genetic Therapy/methods , Lung Neoplasms/secondary , Neovascularization, Pathologic , Protein Precursors/genetics , Adenoviridae/genetics , Analysis of Variance , Animals , Aorta , Carcinoma, Lewis Lung/blood supply , Carcinoma, Lewis Lung/therapy , Cattle , Cells, Cultured , Endothelin-1/genetics , Gene Expression , Gene Targeting/methods , Genetic Vectors/administration & dosage , Green Fluorescent Proteins , Liver/metabolism , Luminescent Proteins/genetics , Lung Neoplasms/blood supply , Lung Neoplasms/therapy , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Microscopy, Phase-Contrast , Promoter Regions, Genetic , Statistics, NonparametricABSTRACT
BACKGROUND: It has been proposed that autoimmune factors can influence the progression of atherosclerosis. We have previously shown that immunization of LDL receptor-deficient (LDL-RD mice) with beta(2)-glycoprotein I (beta2GPI; a principal target of "autoimmune" antiphospholipid antibodies) enhances early atherosclerosis. In the present study, we tested the hypothesis that adoptive transfer of beta2GPI-reactive T cells can accelerate fatty streak formation in LDL-RD mice. METHODS AND RESULTS: LDL-RD mice were immunized with human beta2GPI. An additional group of mice were immunized with beta2GPI and boosted with the same antigen 3 weeks later. Control mice with immunized with human serum albumin. Lymphocytes obtained from the draining lymph node cells or from splenocytes of beta2GPI- or human serum albumin-immunized mice were stimulated in vitro with beta2GPI or with the mitogen concavalin A, respectively. The cultured lymphocytes were transferred intraperitoneally to syngenic LDL-RD mice, and the mice were fed a high-fat "Western" diet for 5 weeks until death. Mice injected with lymphocytes from draining lymph nodes or spleens of beta2GPI-immunized animals displayed larger fatty streaks than those induced by control treated animals. T-cell-depleted splenocytes from beta2GPI were unable to promote lesion formation in the mice. CONCLUSIONS: The present study provides the first direct evidence for a role of antigen (beta2GPI)-reactive T cells in the promotion of fatty streaks in mice.
Subject(s)
Arteriosclerosis/immunology , Glycoproteins/immunology , Receptors, LDL/metabolism , T-Lymphocytes/immunology , Adoptive Transfer , Analysis of Variance , Animals , Antibodies/immunology , Arteriosclerosis/metabolism , Arteriosclerosis/pathology , Cholesterol/metabolism , Cytokines/metabolism , Disease Models, Animal , Disease Progression , Female , Humans , Lymph Nodes/metabolism , Male , Mice , Mice, Inbred C57BL , Receptors, LDL/deficiency , T-Lymphocytes/transplantation , beta 2-Glycoprotein IABSTRACT
Diabetes and atherosclerosis have been proposed to be influenced by immune and autoimmune mechanisms. A common incriminated antigen in both disorders is the heat shock protein (HSP)-60/65. In the current study, we established a model combining hyperglycemia with hyperlipidemia in LDL receptor-deficient (LDL-RD) mice and assessed its possible influences on lipid profile, HSP60/65, and atherogenesis. LDL-RD mice were injected either with streptozotocin to induce hyperglycemia or with citrate buffer (control). When hyperglycemia was induced, both study groups were challenged with a high-fat (Western) diet for 6 weeks. Plasma fasting glucose, lipid profile, and antibody levels to HSP65 and oxidized LDL were assessed. At death, the spleens from both groups were evaluated for their proliferative response to HSP65 and the consequent cytokine production. The extent of atherosclerosis was assessed at the aortic sinus. Plasma glucose, cholesterol, and triglyceride levels were elevated in mice injected with streptozotocin compared with control mice. Atherosclerotic lesions were significantly larger in the streptozotocin-injected hyperglycemic LDL-RD mice (132 +/- 23 x 10(5) microm2) in comparison to their normoglycemic litter-mates (20 +/- 6.6 x 10(5) microm2; P < 0.0001). Both humoral and cellular immune response to HSP65 was more pronounced in streptozotocin-injected mice. When challenged with HSP65 in vitro, splenocytes from streptozotocin-injected mice favored the production of the T-helper (TH)-1 cytokine gamma-interferon. In conclusion, we have established a mouse model that combines hyperglycemia with diet-induced hyperlipidemia in LDL-RD mice and studied its effect on atherosclerosis progression. The accelerated atherosclerotic process is associated with heightened immune response to HSP65 and a shift to a TH1 cytokine profile.
Subject(s)
Arteriosclerosis/complications , Bacterial Proteins , Hyperglycemia/complications , Hyperlipidemias/complications , Receptors, LDL/deficiency , Animals , Antibodies/analysis , Aorta/pathology , Arteriosclerosis/pathology , Blood Glucose/analysis , Body Weight , Chaperonin 60 , Chaperonins/immunology , Cytokines/biosynthesis , Female , Hyperglycemia/blood , Hyperglycemia/chemically induced , Hyperglycemia/immunology , Hyperlipidemias/blood , Hyperlipidemias/chemically induced , Hyperlipidemias/immunology , Immunity , Immunity, Cellular , Lipids/blood , Lipoproteins, LDL/immunology , Male , Mice , Mice, Inbred C57BL , Spleen/immunology , Spleen/metabolism , Spleen/pathology , StreptozocinABSTRACT
Atherosclerotic lesions can be induced in rabbits and mice immunized with heat shock protein 65 (HSP65). In the current study, we investigated the role of interleukin (IL)-4 in the HSP65- and Mycobacterium tuberculosis (MT)-induced models that exhibit an inflammatory phenotype. Fatty streak formation in IL-4-knockout (IL-4 KO) mice immunized with HSP65 or MT was significantly reduced when compared with lesions in wild-type C57BL/6 mice. However, when injected with control (HSP-free) adjuvant, no differences were evident in the lesion size between wild-type and the IL-4 KO mice. Next, we studied comparatively the extent of humoral and cellular immune responses to HSP65 in the IL-4 KO and wild-type mice, as those are thought to be influential in murine atherosclerosis. Anti-HSP65 antibody levels were reduced in the HSP65-immunized IL-4 KO mice as compared with their wild-type littermates, whereas no differences were evident between the groups with respect to the primary cellular immune response to HSP65. Other than the absence of IL-4 in the knockout mice, the pattern of secreting cytokines interferon-gamma and IL-10 in concanavalin A-primed splenocytes was similar between the groups. HSP65-primed inguinal lymphocytes from IL-4 KO mice immunized with HSP65 secreted higher levels of interferon-gamma (previously shown to be proatherogenic in vivo) as compared with their wild-type controls. 12-/15-Lipoxygenase expression, known to be regulated by IL-4 and to contribute to murine atherosclerosis, in the lesions was not influenced by the immunization protocol used or by IL-4 disruption. Thus, IL-4 may prove a principal cytokine in the progression of early "inflammatory" atherosclerotic lesions and may serve as a target for immunomodulation.
Subject(s)
Arteriosclerosis/immunology , Bacterial Proteins , Chaperonins/physiology , Interleukin-4/physiology , Mycobacterium tuberculosis/immunology , Animals , Antibodies/analysis , Arachidonate 12-Lipoxygenase/metabolism , Arachidonate 15-Lipoxygenase/metabolism , Arteriosclerosis/blood , Cell Division , Chaperonin 60 , Chaperonins/immunology , Cholesterol/blood , Female , Immunization , Interferon-gamma/biosynthesis , Interleukin-10/biosynthesis , Interleukin-4/biosynthesis , Interleukin-4/deficiency , Interleukin-4/genetics , Lymphocytes/pathology , Macrophages, Peritoneal/enzymology , Mice , Mice, Inbred C57BL , Mice, Knockout/genetics , Reference ValuesABSTRACT
The modern view of atherosclerosis is of a chronic inflammatory disorder. In accord with this paradigm, the process of uninhibited influx of fat to the vessel wall results from an 'adequate' response to various forms of injury (i.e. turbulence, infections, modified lipoproteins). This idea has been further extended by several groups, to assume that the atherosclerotic lesion can be the target of an autoimmune mediated attack. According to this hypothesis, the site of initiation of the plaque should bear/express the target autoantigen, whereas concomitantly a respective immune response is generated in the periphery. The examples illuminating this notion are beta2GPI as a target autoantigen, HSP60/65 an oxidized-LDL. Herein we present evidence to support the involvement of autoimmune mechanisms in atherogenesis based on the experience from experimental models and human studies.
Subject(s)
Arteriosclerosis/immunology , Autoimmunity , Bacterial Proteins , Animals , Antibodies, Antiphospholipid/metabolism , Arteriosclerosis/etiology , Arteriosclerosis/pathology , Autoantigens , Chaperonin 60/immunology , Chaperonins/immunology , Disease Models, Animal , Glycoproteins/immunology , Humans , Inflammation/etiology , Inflammation/immunology , Lipoproteins, LDL/immunology , Lupus Erythematosus, Systemic/complications , Lupus Erythematosus, Systemic/immunology , beta 2-Glycoprotein IABSTRACT
Heat shock proteins are a family of approximately 25 highly conserved proteins upregulated in response to various forms of stress. They play an active role in the development autoimmune diseases in animals, and have been incriminated in human autoimmune diseases (i.e. rheumatoid arthritis, multiple sclerosis). It has been previously shown, that an induced immune response against Heat shock protein 65 (HSP-65) results in atherosclerotic lesions in normocholesterolemic rabbits. We have supported these findings showing that C57BL/6 mice immunized with HSP-65 and fed a high-fat diet develop enhanced fatty streaks. To create a model that will eliminate the need for exogenous supplementation of a high-fat diet, we have immunized LDL receptor deficient (LDL-RD) mice with HSP-65 or with heat-killed Mycobacterium tuberculosis (Mt). Seven groups of LDL-RD mice (n=10), were immunized subcutaneously with different concentrations of HSP-65, Mt or bovine serum albumin (BSA). All mice were fed a normal chow-diet for 3 months. The mice immunized with the higher doses of Mt developed significantly larger fatty streaks when compared with their BSA immunized littermates. The size of the lesions in the aortic sinus were: 31,562+/-5,994 microm(2)in the 10 microg Mt and 52,777+/-5,245 microm(2)in the 100 microg Mt vs. 11, 500+/-3,750 microm(2)in the BSA group (P<0.05). In the HSP-65-immunized mice, only the group injected with the highest dose (5 microg, twice) developed significantly larger fatty streaks when compared with the BSA-immunized group (28,611+/-4,716 microm(2)vs. 11,500+/-3,750 microm(2)respectively, (P<0.05). The HSP-65-but not the Mt- or BSA-immunized mice developed high titers of anti HSP-65 antibodies, beginning 10 days after the immunization, which persisted until they were killed. Immunohistochemical staining showed CD3-positive lymphocytes in the aortic sinus of mice immunized with Mt or HSP-65, but not in the control group. Thus, we established a mouse model of HSP-65 immune mediated atherosclerosis devoid of high fat diet supplementation. This model will enable us to further study the role of the immune system in atherosclerosis, via HSP-65 and raise novel immunomodulatory therapeutic modalities.
Subject(s)
Arteriosclerosis/etiology , Arteriosclerosis/immunology , Bacterial Proteins , Chaperonins/immunology , Receptors, LDL/deficiency , Animals , Arteriosclerosis/blood , Autoantibodies/blood , Cattle , Chaperonin 60 , Cholesterol/blood , Disease Models, Animal , Female , Humans , Immunization , Lipoproteins, LDL/blood , Lymphocyte Activation , Lymphocytes/immunology , Lymphocytes/pathology , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Rabbits , Serum Albumin, Bovine/immunology , Sinus of Valsalva/pathologyABSTRACT
Complement components in breast milk may enhance the local immune response in the gut of infants. In this study, we investigated the expression of complement genes in the mammary gland and attempted to determine possible regulatory mechanisms. We have studied the expression of C3, C4, factor B, and HLA-DRalpha mRNA by in situ hybridization in gestational mammary gland specimens and compared these findings to those in breast tissue affected with an inflammatory process, lactating adenoma or idiopathic gynecomastia. In normal resting breast, only C4 mRNA was noted in some ductal epithelium. In gestational mammary gland, there was a diffuse expression of C4, C3, and factor B mRNA in the epithelial cells of the acini. A similar pattern of complement gene expression was found in localized areas of an infectious inflammatory process. In addition, in the inflammatory specimens, there was also expression of C3 mRNA in infiltrating macrophages (CD 68 positive cells). In gynecomastia, C4 mRNA was noted in ductal epithelium, and there was a marked increased expression of C3 mRNA in the proliferating epithelium of the lactating adenoma. HLA-DRalpha was observed only in macrophages involved in the inflammatory response. Our findings, which reflect the hormonal and inflammatory events in vivo, provide new insights as to in situ complement gene expression.
Subject(s)
Breast/metabolism , Complement System Proteins/genetics , Mastitis/metabolism , Pregnancy Complications , Adenoma/metabolism , Adenoma/pathology , Adult , Biopsy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Complement C3/genetics , Complement C4/genetics , Complement Factor B/genetics , Female , Gene Expression , Gynecomastia/metabolism , Gynecomastia/pathology , Humans , Lactation/metabolism , Male , Middle Aged , Pregnancy , Pregnancy Complications, NeoplasticABSTRACT
The thymus reaches its maximal size at the age of 1 month in ICR mice and thereafter, the thymic cortex undergoes an exponential decline. This study was designed to compare the proliferation and apoptosis of thymocytes in different parts of the thymus of ICR female mice at the beginning and after the rapid phase of decline of the thymic cortical cellularity. The pattern of proliferation and apoptosis of the thymus was studied in situ in 1-month-old ICR female mice (10 mice) compared to mice at 7 months of age (10 mice). Staining for argyrophylic nucleolar organizer region by histochemistry was used to determine the proportion of type 2 thymocytes, which are considered as cells at S phase of the cell cycle. The mean number of type 2 cells in four random samples of 50 cells in each part of the thymus was defined as the proliferation index of this part of the thymus. In situ detection of apoptosis of thymocytes was carried out using the Apoptag kit, which can detect a single cell apoptosis. The mean number of apoptotic cells in five randomly selected fields of each part of the thymus was defined as the apoptotic index of this part of the thymus. The proliferation index of the peripheral cortex of the 1-month-old mice was 3.6 times higher than the proliferation index of the deep cortex and 5.8 times higher than the proliferation index of the medulla (P < 0.0001). The proliferation index of the peripheral cortex of the 7-month-old mice was reduced by 45% compared to the 1-month-old mice (P < 0.005). The apoptotic index of the corticomedullary junction of the 1-month-old mice was six times higher than the apoptotic index of the cortex and 18 times higher than the apoptotic index of the medulla. The apoptotic index of the thymic cortex was elevated by 66% in the 7-month-old mice compared to the 1-month-old mice (P < 0.0001). We conclude that there is a reduction of the proliferation index and an elevation of the apoptotic index of the thymic cortex in adult mice compared to young mice. These changes might account for the reduction of thymic cortical cellularity during thymic involution.