The pathogenesis of microcephaly resulting from congenital infections: why is my baby's head so small?

The emergence of Zika-virus-associated congenital microcephaly has engendered renewed interest in the pathogenesis of microcephaly induced by infectious agents. Three of the original "TORCH" agents are associated with an appreciable incidence of congenital microcephaly: cytomegalovirus, rubella virus, and Toxoplasma gondii. The pathology of congenital microcephaly is characterized by neurotropic infectious agents that involve the fetal nervous system, leading to brain destruction with calcifications, microcephaly, sensorineural hearing loss, and ophthalmologic abnormalities. The inflammatory reaction induced by these four agents has an important role in pathogenesis. The potential role of "strain differences" in pathogenesis of microcephaly by these four pathogens is examined. Specific epidemiologic factors, such as first and early second trimester maternal infection, and the manifestations of congenital infection in the infant, shed some light on the pathogenesis. Immune aspects of normal pregnancy and their role in congenital infections is examined. In this review, we integrate all these findings to create a unified hypothesis of the pathogenesis of congenital microcephaly induced by these infectious agents.

The possible role of NS3 protease activity of hepatitis C virus on fibrogenesis and miR-122 expression in hepatic stellate cells.

The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors. The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression. However, the possible correlation between miR-122 levels and fibrosis state has been less investigated. Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line. Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1). Moreover, the transforming growth factor beta (TGF-ß) levels in the supernatants of transfected cells were evaluated by ELISA. The gene expression analysis of fibrotic genes and TGF-ß cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001). The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function. These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122.

Molecular epidemiology of hepatitis B virus in Iran.

Hepatitis B virus (HBV) infection is a major cause of liver disease worldwide. Eight genotypes and 24 subgenotypes of HBV have been identified. The aim of this study was to determine the distribution of HBV genotypes, subgenotypes and subtypes, and to understand HBV genetic variability in the HBV genome circulating in Iranian provinces. Two hundred and forty-nine sera from HBV-infected patients living in 25 provinces of Iran were collected (2004-2007). A part of the HBV S/pol and whole BCP/C genes were amplified, sequenced and then subjected to phylogenetic, recombination and genetic variability analysis. Results revealed genotype D of HBV in all samples and subgenotypes D1 (98.52%), D2 (0.74%) and D3 (0.74%) among Iranian patients living in different provinces of Iran. Subtypes ayw2 (94.4%), ayw1 (2.8%), ayw3 (2%) and ayw4 (0.4%) were deduced, on the basis of HBV small surface antigen (HBsAg) amino acid sequences. The mean percentage intra-genotypic distance of S plus core regions was 2.8%; the mean percentage inter-genotypic distance of this region between Iranian strains and genotype D isolates was 3.1%; and this rate for other genotypes was 5.2-11.4%. Various rates of point mutations have been found within different HBV genes, e.g. HBsAg (17.2%), precore-G1896A (59.5%) and Basal core promoter (BCP) double mutations (49.2%), whereas no recombination was found. In conclusion, these results indicate that the only genotype circulating in the provinces of Iran is genotype D. There exist high genetic variabilities in the S/pol and BCP/C regions among the Iranian HBV isolates.

Antigen-specific lymphocyte proliferation assay and virus neutralization test for measurement of measles-specific immunity in 15-19 years old high school students in Tehran, Iran.

Limited information is available concerning the role of measles-specific cell mediated immunity as a correlate of long-term protection from measles infection. Although serological responses are determined in epidemiological studies and high antibody titer is a good indicator of protection, the role of Cell-Mediated Immunity (CMI) has to be defined more clearly. In this study, Lymphocyte Proliferation (LP) and Viral Neutralization Test (VNT) were used in order to measure measles-specific cellular and humoral immune responses of 100 high school students in Tehran. From total number of subjects studied, 33 were girls and 67 were boys and all were in good health. Of these, 77 had protective neutralizing measles antibody titers and 23 did not have such titer. The results of LP showed that 89 subjects had protective cellular immune responses and 11 did not. A quantitative relationship between humoral and cellular immune responses was not observed. These findings suggest that measles-specific protective CMI is measurable for longer time in comparison to humoral immunity. These data suggest that LP responses may be better sustained than antibody titers in some children.

Improvement in isolation of human peripheral blood leukocyte subpopulations: application in diagnosing human cytomegalovirus infection in bone marrow transplant patients.

OBJECTIVES: High-yield isolation and purification of human leukocyte subpopulations from whole blood is fundamental to many biological and medical applications including qualitative and quantitative PCR-based techniques of determining human cytomegalovirus infection. Several procedures have been reported to purify morphologically and functionally intact human leukocyte subpopulations for diagnostic proposes. Here, we report and evaluate a technique for high-yield purification of intact and viable human leukocyte subpopulations based on modification of a previous methodology. MATERIALS AND METHODS: One hundred peripheral blood samples were collected from bone marrow transplant recipients (n = 60), bone marrow donors (n = 20), and healthy blood donors (n = 20). The samples were tested in parallel using 4 different leukocyte separation methods. The methods were evaluated based on the concentration, purity, and viability of the isolated leukocyte subpopulations. RESULTS: When compared with standard methods, our methods produced 99% purity for both polymorphonuclear or mononuclear leukocytes. The corresponding viability for the methods was determined to be 98%. No erythrocyte contamination was demonstrated. However, the maximum concentration for polymorphonuclear or mononuclear leukocytes obtained by standard methods was 70%. The corresponding viability for all the methods was determined to be 98%. CONCLUSIONS: Our results indicate that in patients with decreased whole blood leukocyte numbers, using either a modified Ficoll NH(4)Cl or a modified dextran method would be valuable for simultaneous separation of polymorphonuclear and mononuclear leukocytes with high purity, viability, and concentration.

Comparative analysis of a double primer PCR assay with plasma, leukocytes and antigenemia for diagnosis of active human cytomegalovirus infection in bone marrow transplant patients.

The aim of the study was to determine the prognostic value of a double primer PCR assay to detect human cytomegalovirus (HCMV) infection or disease in bone marrow transplant (BMT) recipients. A total of 209 blood samples including peripheral blood mononuclear cells (PBMN), polymorphonuclear (PMN) leukocytes and plasma from 26 BMT recipients were tested by PCR assay. To discriminate between latent and active HCMV infection, 177 blood samples were also tested by a quantitative antigenemia assay. HCMV serology status of donors and recipients was determined before transplantation by an enzyme immunosorbent assay method. Using the double primer PCR assay, the number of positive samples increased by an average of 11.6%. Symptomatic active HCMV infection was diagnosed in 14 (53.8%) out of 26 BMT patients. There was a good association between double primer PCR assay of PMN leukocytes and antigenemia assays for detection of active HCMV infection in all patients. Detection of HCMV DNA in PMN leukocytes of BMT patients by double primer PCR assay can be an alternative method for antigenemia assay. However, quantitative PCR methods will be necessary for monitoring antiviral treatment.

Role of rotaviruses in children with acute diarrhea in Tehran, Iran.

BACKGROUND: Rotavirus illness is associated with significant cause of morbidity and is a common cause of hospitalization worldwide. OBJECTIVE: This study was performed to assess the role of rotaviruses in children presenting with acute diarrhea in two main Children's Medical Centers and one general hospital in Tehran. STUDY DESIGN: Stool specimens from 704 children less than 5 years of age suffering from diarrhea were tested for the presence of rotaviruses by a monoclonal antibody-based enzyme immunoassay. A total of 176 fecal specimens collected from healthy children in similar age group were studied as controls. RESULTS: Rotavirus antigen was detected in 15.3% of patients. Infants between 6 and 12 months of age were most frequently affected. Rotavirus infection was significantly less frequent in breast-fed than among bottle-fed babies. Watery diarrhea was present in 68.5% of children. Detection rate was highest in the spring and lowest in summer. Rotavirus can be regarded as a major etiologic agent of acute diarrhea in infants and children up to 5-years-old in Iran, immunization at birth may protect the children before their first symptomatic infection.

Development of an enzyme-linked immunosorbent assay for immunoglobulin M antibodies against measles virus.

Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and then an anti-IgM antibody was produced in rabbits and purified in a multistep process. The rabbit IgG against human IgM was conjugated with peroxidase. Measles virus-infected Vero cells produced viral antigen. One hundred serum samples from infants of 9 to 18 months of age, mostly vaccinated, were evaluated for determining the presence of specific IgM antibodies against measles virus. The samples were also evaluated for neutralizing antibodies against measles virus by a microneutralization test (MNT). By comparing the results of the ELISA with those of MNT, it was demonstrated that ELISA had a sensitivity and specificity of 100 and 92%, respectively. On the other hand, when the results obtained by our ELISA system were compared with those of an imported measles virus IgM ELISA kit (EIAgen; Adaltis Italia SPa, Bologna, Italy), a high level of agreement was shown (k = 0.926).

Incidence of enteric adenovirus gastroenteritis in Iranian children.

BACKGROUND: Enteric adenoviruses, i.e. adenovirus 40 (Ad40) and adenovirus 41 (Ad41), have been shown to be a substantial cause of pediatric gastroenteritis in various parts of the world, but no data are available for Iran. OBJECTIVE: The present study was performed to determine the incidence of enteric adenoviruses in children presenting to the Children's Medical Center with gastroenteritis in Iran. STUDY DESIGN: Stool specimens from 872 children less than 7 years of age attending the Children's Medical Center in Tehran, Iran, with gastroenteritis were tested for the presence of Ad40, Ad41, and adenovirus-genus by a monoclonal antibody-based enzyme immunoassay. RESULTS AND CONCLUSION: 6.7% of stool specimens contained enteric adenoviruses (3.3% Ad40 and 3.4% Ad41) and 2.0% nonenteric adenoviruses. Mean ages of Ad40, Ad41 and NEAd-positive children were 21, 19 and 29 months, respectively. Among the adenovirus-positive patients, 53.9% were male and 46.1% female. Watery diarrhea was present in 86.4% of children infected by adenoviruses. In conclusion, for the first time, we demonstrated the presence of enteric and nonenteric adenoviruses in a considerable proportion of stool samples from Iranian children with gastroenteritis.

Antibodies to enteric adenoviruses (Ad40 and Ad41) in sera from Iranian children.

BACKGROUND: There is strong epidemiologic and serologic evidence that infection with the enteric adenoviruses can result in severe gastroenteritis in children. OBJECTIVES: This study was performed to determine the prevalence of enteric adenovirus infection in Iran. STUDY DESIGN: One hundred and twenty-seven single sera from children up to 7 years of age, collected from healthy Iranian children in 1993-1994, were tested for antibodies to enteric adenoviruses by neutralization tests. RESULT AND CONCLUSION: Antibodies to enteric adenoviruses have been detected in about one-half of sera. It is concluded that infection by these viruses is common among children in Iran.

Qualitative and quantitative analysis of T lymphocytes during normal human pregnancy.

PROBLEM: Human reproduction involves contact between cells which are allogeneic to one another, however the fetus not only survives but thrives. METHODS: Aspects of T-cell-mediated immunity during normal human pregnancy were studied. PBMNCs of pregnant and nonpregnant women were stimulated with PHA and cytomegalovirus antigens (CMV). The capacity of stimulated cells to proliferate, to produce IL-2 and IFN-gamma, to express IL-2 receptor (IL2R1) and the effect of rIL2 on the proliferation rate of lymphocytes were examined. FACS was utilized for T-cell subset comparisons. RESULTS: The proliferation rate, IL-2, and IFN-gamma synthesis were all significantly impaired at suboptimal concentration of PHA throughout pregnancy. Exogenous rIL-2 corrected this depression of cell-mediated immunity (CMI). At optimal concentration of PHA, proliferation rate and production of IFN-gamma and IL-2 were all decreased. Exogenous rIL-2 corrected these deficits only in the third trimester. Third trimester pregnant women demonstrated a significant depression of proliferation as well as IL-2 and IFN-gamma production after CMV stimulation, which was partially corrected by exogenous rIL-2. FACS analysis suggested that after stimulation by CMV and optimal concentration of PHA, T cells were activated and both CD4+ and CD8+ lymphoblasts expressed normal density of IL-2R1. With suboptimal PHA, the number of activated CD4+ and CD4+IL2R1+ cells were diminished and CD4+ and CD8+ T lymphoblasts expressed lower number of IL2R1. CONCLUSIONS: CD4 T helper (Th1) cell function is down regulated progressively during the three trimesters of pregnancy without changes in the quantity of T cell subsets.