Males and females differ in their immunological responses to foreign and self-antigens and show distinctions in innate and adaptive immune responses. Certain immunological sex differences are present throughout life, whereas others are only apparent after puberty and before reproductive senescence, suggesting that both genes and hormones are involved. Furthermore, early environmental exposures influence the microbiome and have sex-dependent effects on immune function. Importantly, these sex-based immunological differences contribute to variations in the incidence of autoimmune diseases and malignancies, susceptibility to infectious diseases and responses to vaccines in males and females. Here, we discuss these differences and emphasize that sex is a biological variable that should be considered in immunological studies.

/pdf/sex-differences-in-immune-responses-5lxzp3yqfg.pdf

Sex differences in immune responses

Factors that inhibit the amplification of nucleic acids by PCR are present with target DNAs from many sources. The inhibitors generally act at one or more of three essential points in the reaction in the following ways: they interfere with the cell lysis necessary for extraction of DNA, they interfere by nucleic acid degradation or capture, and they inhibit polymerase activity for amplification of target DNA. Although a wide range of inhibitors is reported, the identities and modes of action of many remain unclear. These effects may have important implications for clinical and public health investigations, especially if the investigations involve food and environmental screening. Common inhibitors include various components of body fluids and reagents encountered in clinical and forensic science (e.g., hemoglobin, urea, and heparin), food constituents (e.g., organic and phenolic compounds, glycogen, fats, and Ca 21 ), and environmental compounds (e.g., phenolic compounds, humic acids, and heavy metals). Other, more widespread inhibitors include constituents of bacterial cells, nontarget DNA and contaminants, and laboratory items such as pollen, glove powder, laboratory plasticware, and cellulose. This review discusses the findings of many studies related to clinical, food, and environmental microbiology, including approaches that have been used to overcome inhibition and facilitate amplification for detection and typing. Few areas of biological science remain untouched by the invention of PCR (34, 81, 99). Other methods for amplifying nucleic acids (72, 123), such as Qb replicase (18), ligase chain reaction (13, 128), single-stranded sequence replication (17, 47), strand displacement amplification (126, 127), and nucleic acid sequence-based amplification (23, 122), have been described, but these methods have received less attention. Problems sometimes occur with PCR, however (124). Despite early indications of great sensitivity, the sensitivity of PCR may be a negative aspect of the procedure, since the most commonly reported problem is false-positive results due to cross-contamination (98, 124). This problem can be overcome by UV irradiation (100), with sodium hypochlorite (92), and by photochemical or enzymic methods (25, 36, 40, 78). One problem that is less discussed is reaction inhibition. This may be total or partial and can manifest itself as complete reaction failure or as reduced sensitivity of detection. In some cases, inhibition may be the cause of false-negative reactions, since few workers incorporate internal controls in each reaction tube. Early evidence of exquisite sensitivity with mammalian cells (53) involving detection of a single molecule of DNA from a hair was not reproduced when PCR was applied to many microbial (and some mammalian) situations, where poor sensitivity, specificity, and reproducibility have been reported (16, 82, 86, 129, 132, 134). There may also be potentially important effects in PCR typing reactions (121), and difficulties can occur in post-PCR manipulation (61). Although systematic study of inhibition has seldom been the focus of published investigations, many workers have reported these effects in the course of other studies (12, 19, 21, 124, 129, 132, 133). Considering the prevalence of this problem, it is surprising that few systematic and mechanistic studies of PCR inhibition have been reported. Rossen et al. (97) contributed the most comprehensive study of PCR inhibition, identifying inhibitory factors in foods, bacterial culture media, and various chemical compounds. These inhibitory factors included organic and inorganic chemicals, detergents, antibiotics, buffers, enzymes, polysaccharides, fats, and proteins. This review lists and discusses inhibitors and methods that can overcome the attenuation of amplification in clinical, food, and environmental microbiology. It is beyond the scope of this paper to discuss in detail the various physical, enzymic, and chemical methods used in the extraction, purification, and quantitation of nucleic acids. Those methods are presented and discussed in commercial literature and elsewhere (14, 95, 96, 106, 134).

https://aem.asm.org/content/aem/63/10/3741.full.pdf

Inhibition and facilitation of nucleic acid amplification.

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

In most PCR-based tissue typing techniques the PCR amplification is followed by a post-amplification specificity step. In typing by PCR amplification with sequence-specific primers (PCR-SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR "low-resolution" typing by PCR-SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1-DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1-DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR "low-resolution" PCR-SSP technique, TaqI DRB-DQA-DQB RFLP analysis and serology. The concordance between PCR-SSP typing and RFLP analysis was 100%. The reproducibility was 100% in 40 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained. All homozygous and heterozygous combinations of DR1-DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post-amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR-SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR "low-resolution" typing by the PCR-SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor-recipient matching in cadaveric transplantations.

HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation.

A novel form of Epstein-Barr virus latency in normal B cells in vivo.

IFN-α exercises multiple immune modulatory and antiviral activities and has been suggested to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Plasmacytoid dendritic cells (pDCs) release IFN-α upon TLR7 and TLR9 ligation. With respect to the nine times higher incidence of SLE in women and the clinical use of synthetic TLR ligands as novel immune adjuvants, we analyzed IFN-α and TNF-α production in healthy human individuals. Blood samples were incubated with synthetic TLR7 and TLR9 ligands. In three independent groups ( n 1 = 120, n 2 = 101, and n 3 = 123), analysis revealed a capacity of female PBLs to produce significantly higher IFN-α levels after TLR7 stimulation ( p 1 p 2 p 3 TLR7 gene was investigated in monoclonal B cell lines and, independently, in pDCs after cell sorting and single-cell picking, indicating regular silencing of one TLR7 allele in females. Additionally, exogenous 17β-estrogen and estrogen receptor antagonism did not indicate a significant role on TLR7-induced IFN-α production. Our data reveal for the first time a profound sex-dependent pathway of TLR7-induced IFN-α with higher production in females. These findings may explain the higher prevalence of SLE in females and the reported decreased therapeutic efficacy of synthetic TLR7 ligands in male individuals.

TLR7 Ligands Induce Higher IFN-α Production in Females

Cytomegalovirus (CMV) infection is an important cause of disease in immunocompromised patients. In a prospective longitudinal study of 34 septic patients, the incidence of active CMV infection was examined. Eleven of 34 patients (32.4%) had active CMV infection, diagnosed by immunocytochemical staining of CMV pp65 antigen in blood leukocytes and/or detection of CMV DNA by PCR. Positive results for CMV infection were obtained in a median of 4 days (by PCR) or 11 days (by staining of pp65 antigen) after onset of sepsis. Twenty patients for whom more than one sample was examined were selected for further analysis. Among the patients with active CMV infection (nine of 20) there was a trend toward higher median values of tumor necrosis factor alpha, interleukin-1 beta, alanine aminotransferase, and aspartate aminotransferase in plasma, in comparison with the values for patients without CMV infection. Sepsis in patients with CMV infection may affect outcome of the disease.

High Incidence of Active Cytomegalovirus Infection Among Septic Patients

State of the art genotyping of HLA class II alleles with group-specific DNA amplification by the polymerase chain reaction (PCR) (1) and subsequent probing with sequence-specific oligonucleotides (2–4) is not suitable for typing cadaveric organ donors since the typing procedure takes far more than one working day. We designed specific oligonucleotide primer sets for nested PCR amplification which allowed typing for all serological HLA-DR specificities (DR1-DRw18) solely by the detection of amplified DNA in the reaction mixtures after agarose gel electrophoresis. Exon 2 of the DRB genes and a DRw52-group-specific part of DRB1 exon 2 was amplified directly from cell lysates without prior DNA extraction. The amplified DNA was subjected to a second round of amplification, which employed a set of 18 nested allele- or group-specific primer pairs. All alleles which have at least a single mismatched base at the terminal 3′-nucleotide of one primer were completely refractory to amplification. This assay is easy to perform and takes less than one working day to complete. Thus, this method may prove to be suitable for DNA typing of organ donors for prospective HLA-DR matching in renal transplantation.

Rapid HLA‐DRB1 genotyping by nested PCR amplification

We designed a highly sensitive and specific polymerase chain reaction assay for the detection of Epstein-Barr virus (EBV)-related sequences in B-cell DNA of EBV-seropositive healthy individuals. By using this assay, we were able to amplify at least 10 copies of a plasmid containing the BamHI-W region, which is repeated up to 11 times within the EBV genome, in the presence of 1 microgram of EBV-negative DNA, indicating that one virus genome was detectable from 150,000 cells. In 15 of 16 tested individuals, EBV-related sequences were found frequently when the DNA from 10(6) B lymphocytes was examined and 1 microgram of DNA was used in each polymerase chain reaction. When the results of amplifying the diluted plasmid were used as a semiquantitative standard, the number of EBV genomes detected could be estimated to range between 50 and less than 1 from 10(6) B lymphocytes. The results of our study will provide the basis for further investigations of the characteristics of the latent carrier state in healthy EBV-seropositive individuals, such as the determination of the number of virus copies per cell and expression of antigens.

Detection and quantification of latently infected B lymphocytes in Epstein-Barr virus-seropositive, healthy individuals by polymerase chain reaction.

Typing for human platelet antigens (HPA) is useful in a variety of clinical situations. We developed a method for genotyping for HPA-1, -2, -3 and -5 by means of the PCR amplification with sequence-specific primers (PCR-SSP) technique. Primer sets were designed to allow PCR amplification for all systems using the same assay conditions. Specificity and sensitivity of the method were assessed in a blind quality control study (n = 112). In 111 cases, results obtained by PCR-SSP were identical as compared with PCR-restriction fragment length polymorphism technique. One discrepancy was found to be due to a typing error in the data sheet. The results of the PCR-SSP technique were available within 3 h. We conclude that genotyping based on PCR-SSP enables rapid typing for HPA systems, which makes this technique feasible in most clinical settings where urgent HPA typing is required.

Gregor Bein

Papers

TLR7 Ligands Induce Higher IFN-α Production in Females

High Incidence of Active Cytomegalovirus Infection Among Septic Patients

Rapid HLA‐DRB1 genotyping by nested PCR amplification

Detection and quantification of latently infected B lymphocytes in Epstein-Barr virus-seropositive, healthy individuals by polymerase chain reaction.

Rapid typing for human platelet antigen systems-1, -2, -3 and -5 by PCR amplification with sequence-specific primers.