Evaluation of in vitro SARS-CoV-2 inactivation by a new quaternary ammonium compound: Bromiphen bromide.

The pneumonia (COVID-19) outbreak caused by the novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which unpredictably exploded in late December of 2019 has stressed the importance of being able to control potential pathogens with the aim of limiting their spread. Although vaccines are well known as a powerful tool for ensuring public health and controlling the pandemic, disinfection and hygiene habits remain crucial to prevent infection from spreading and maintain the barrier, especially when the microorganism can persist and survive on textiles, surfaces, and medical devices. During the coronavirus disease pandemic, around half of the disinfectants authorized by the US Environmental Protection Agency contained quaternary ammonium compounds (QACs); their effectiveness had not been proven. Herein, the in vitro SARS-CoV-2 inactivation by p-bromodomiphen bromide, namely bromiphen (BRO), a new, potent, and fast-acting QAC is reported. This study demonstrates that BRO, with a dose as low as 0.02%, can completely inhibit SARS-CoV-2 replication in just 30 s. Its virucidal activity was 10- and 100-fold more robust compared to other commercially available QACs, namely domiphen bromide and benzalkonium chloride. The critical micellar concentration and the molecular lipophilicity potential surface area support the relevance of the lipophilic nature of these molecules for their activity.

Upregulation of inflammasome activity and increased gut permeability are associated with obesity in children and adolescents.

BACKGROUND: Immune activation contributes to the persistent state of inflammation associated with metabolic dysfunction in obesity. The specific immune receptors that sense metabolic stress signals and trigger inflammation are nevertheless largely unknown, and little is known on inflammatory and immune gene regulation in obesity. METHODS: The study includes a cross-sectional and a longitudinal arm. Forty children and adolescents were enrolled: 22 obese subjects and 18 age-matched normal weight controls. Obese subjects participated in an 18-month therapeutic protocol, based on intensive lifestyle modification (dietary regimen, physical activity and behavioral interventions). Expression of genes involved in the inflammasome pathway, plasma concentration of the inflammasome-associated pro-inflammatory cytokines (interleukin (IL)-1ß and IL-18) and indexes of microbial translocation (lipopolysaccharide (LPS), soluble CD14 (sCD14) and intestinal fatty acid-binding protein) were analyzed at baseline in obese subjects compared with controls, and after 18 months in obese subjects. RESULTS: Cross-sectional analyses showed that the LPS-induced expression of genes involved in inflammasome (NLRP3, caspase 5 and NAIP), Nod-like receptors (NLRX1 and NOD1), downstream signaling (P2RX7, RAGE, RIPk2, TIRAP and BIRC2) and effector molecules (IFN-γ, IL-12ß, IL-1ß, CCL2, CCL5, IL-6 and TNFα) was significantly increased in obese subjects at baseline as compared with normal weight controls. The baseline plasma concentration of inflammasome-related cytokines (IL-1ß and IL-18) and of microbial translocation markers (LPS and sCD14) was augmented in obese subjects as compared with controls as well. Longitudinal analyses indicated that intensive lifestyle modification resulted in a normalization of parameters in subjects with a significant reduction of BMI after 18 months. CONCLUSIONS: In children and adolescents, obesity is characterized by the activation of the inflammasome and by an alteration of gut permeability. Successful lifestyle modification is effective in reducing inflammation, suggesting that inhibition of the inflammasome may be a potential therapeutic strategy in obesity.

Association of complement receptor 2 polymorphisms with innate resistance to HIV-1 infection.

HIV-1 induces activation of complement through the classical and lectin pathways. However, the virus incorporates several membrane-bound or soluble regulators of complement activation (RCA) that inactivate complement. HIV-1 can also use the complement receptors (CRs) for complement-mediated antibody-dependent enhancement of infection (C-ADE). We hypothesize that hypofunctional polymorphisms in RCA or CRs may protect from HIV-1 infection. For this purpose, 139 SNPs located in 19 RCA and CRs genes were genotyped in a population of 201 Spanish HIV-1-exposed seronegative individuals (HESN) and 250 HIV-1-infected patients. Two SNPs were associated with infection susceptibility, rs1567190 in CR2 (odds ratio (OR) = 2.27, P = 1 × 10(-4)) and rs2842704 in C4BPA (OR = 2.11, P = 2 × 10(-4)). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. Although not significant (P = 0.25, OR = 1.57), similar genotypic proportions were obtained for the CR2 marker rs1567190. The results of the two association analyses were combined through a random effect meta-analysis, with a significant P-value of 2.6 x 10(-5) (OR = 2.07). Furthermore, we found that the protective CR2 genotype is correlated with lower levels CR2 mRNA as well as differences in the ratio of the long and short CR2 isoforms.

Immunomodulating activity of Pidotimod in children with Down syndrome.

Acute respiratory tract infections (ARTIs) are the most frequent illnesses in pediatric age, frequently experienced in children with Down Syndrome (DS) due to the associated immune defects of both specific and non-specific immunity. Pidotimod, a synthetic immunostimulant, was shown to reduce the rates of ARTIs in children with DS, however the mechanisms associated with this effect is currently unknown. We analyzed immune parameters in DS children who received the seasonal 2011–2012 virosomal-adjuvanted influenza vaccine. Eighteen children aged 3-10 years (mean age 7.1+/-2.6 years) were randomly assigned (1:1 ratio) to receive Pidotimod 400 mg, administered orally once a day for 90 days or placebo. At the recruitment (T0) all children received a single dose of virosomal-adjuvanted influenza vaccine (Flu). Blood samples were collected at T0 and 3 months after the recruitment (T3) in order to evaluate innate and adaptative immune responses pathway. Flu-specific IgG1 and IgG3 levels in plasma samples were determined at pre-vaccination (T0), and 1 (T1) and 3 months (T3) post-vaccination. The use of Pidotimod was associated with the upregulation of a number of genes involved in the activation of innate immune responses and in antimicrobial activity. Interestingly the ratio of Flu-specific IgG1/IgG3 was skewed in pidotimod-treated individuals, suggesting a preferential activation of complement-dependent effector mechanisms. Although preliminary these data suggest that Pidotimod can potentiate the beneficial effect of immunization, possibly resulting in a stronger activity of both innate and adaptive immune responses.

The pathophysiological role of estrogens in the initial stages of pregnancy: molecular mechanisms and clinical implications for pregnancy outcome from the periconceptional period to end of the first trimester.

BACKGROUND: Estrogens regulate disparate female physiological processes, thus ensuring reproduction. Altered estrogen levels and signaling have been associated with increased risks of pregnancy failure and complications, including hypertensive disorders and low birthweight babies. However, the role of estrogens in the periconceptional period and early pregnancy is still understudied. OBJECTIVE AND RATIONALE: This review aims to summarize the current evidence on the role of maternal estrogens during the periconceptional period and the first trimester of pregnancies conceived naturally and following ART. Detailed molecular mechanisms and related clinical impacts are extensively described. SEARCH METHODS: Data for this narrative review were independently identified by seven researchers on Pubmed and Embase databases. The following keywords were selected: 'estrogens' OR 'estrogen level(s)' OR 'serum estradiol' OR 'estradiol/estrogen concentration', AND 'early pregnancy' OR 'first trimester of pregnancy' OR 'preconceptional period' OR 'ART' OR 'In Vitro Fertilization (IVF)' OR 'Embryo Transfer' OR 'Frozen Embryo Transfer' OR 'oocyte donation' OR 'egg donation' OR 'miscarriage' OR 'pregnancy outcome' OR 'endometrium'. OUTCOMES: During the periconceptional period (defined here as the critical time window starting 1 month before conception), estrogens play a crucial role in endometrial receptivity, through the activation of paracrine/autocrine signaling. A derailed estrogenic milieu within this period seems to be detrimental both in natural and ART-conceived pregnancies. Low estrogen levels are associated with non-conception cycles in natural pregnancies. On the other hand, excessive supraphysiologic estrogen concentrations at time of the LH peak correlate with lower live birth rates and higher risks of pregnancy complications. In early pregnancy, estrogen plays a massive role in placentation mainly by modulating angiogenic factor expression-and in the development of an immune-tolerant uterine micro-environment by remodeling the function of uterine natural killer and T-helper cells. Lower estrogen levels are thought to trigger abnormal placentation in naturally conceived pregnancies, whereas an estrogen excess seems to worsen pregnancy development and outcomes. WIDER IMPLICATIONS: Most current evidence available endorses a relation between periconceptional and first trimester estrogen levels and pregnancy outcomes, further depicting an optimal concentration range to optimize pregnancy success. However, how estrogens co-operate with other factors in order to maintain a fine balance between local tolerance towards the developing fetus and immune responses to pathogens remains elusive. Further studies are highly warranted, also aiming to identify the determinants of estrogen response and biomarkers for personalized estrogen administration regimens in ART.

HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals.

HIV-specific mucosal and cellular immunity was analyzed in heterosexual couples discordant for HIV status in serum and in HIV-unexposed controls. HIV-specific IgA but not IgG was present in urine and vaginal wash samples from HIV-exposed seronegative individuals (ESN), whereas both IgA and IgG were observed in their HIV-seropositive partners; antibodies were not detected in low-risk controls. Envelope protein (Env) peptide-stimulated interleukin-2 (IL-2) production by peripheral blood mononuclear cells (PBMCs) was detected in 9 out of 16 ESNs, 5 out of 16 HIV-infected patients and 1 out of 50 controls. Env peptide-stimulated PBMCs of ESNs produced more IL-2 and less IL-10 compared with those of HIV-infected individuals; no differences were observed in chemokine production or in CCR5 expression. These data demonstrate that a compartmentalized immune response to pathogens is possible in humans and raise the possibility of protective roles for cell-mediated immunity and mucosal IgA in HIV-seronegative individuals exposed to HIV.

SARS-CoV-2 mRNA vaccine BNT162b2 triggers a consistent cross-variant humoral and cellular response.

As the SARS-CoV-2 pandemic continues to rage worldwide, the emergence of numerous variants of concern (VOC) represents a challenge for the vaccinal protective efficacy and the reliability of commercially available high-throughput immunoassays. Our study demonstrates the administration of two doses of the BNT162b2 vaccine that elicited a robust SARS-CoV-2-specific immune response which was assessed up to 3 months after full vaccination in a cohort of 37 health care workers (HCWs). SARS-CoV-2-specific antibody response, evaluated by four commercially available chemiluminescence immunoassays (CLIA), was qualitatively consistent with the results provided by the gold-standard in vitro neutralization assay (NTA). However, we could not observe a correlation between the quantity of the antibody detected by CLIA assays and their neutralizing activity tested by NTA. Almost all subjects developed a SARS-CoV-2-specific T-cell response. Moreover, vaccinated HCWs developed a similar protective neutralizing antibodies response against the EU (B.1), Alpha (B.1.1.7), Gamma (P.1), and Eta (B.1.525) SARS-CoV-2 variants, while Beta (B.1.351) and Delta (B.1.617.2) strains displayed a consistent partial immune evasion. These results underline the importance of a solid vaccine-elicited immune response and a robust antibody titre. We believe that these relevant results should be taken into consideration in the definition of future vaccinal strategies.

Long-term balancing selection maintains trans-specific polymorphisms in the human TRIM5 gene.

The human TRIM5 genes encodes a retroviral restriction factor (TRIM5α). Evolutionary analyses of this gene in mammals have revealed a complex and multifaceted scenario, suggesting that TRIM5 has been the target of exceptionally strong selective pressures, possibly exerted by recurrent waves of retroviral infections. TRIM5 displays inter-individual expression variability in humans and high levels of TRIM5 mRNA have been associated with a reduced risk of HIV-1 infection. We resequenced TRIM5 in chimpanzees and identified two polymorphisms in intron 1 that are shared with humans. Analysis of the gene region encompassing the two trans-specific variants in human populations identified exceptional nucleotide diversity levels and an excess of polymorphism compared to fixed divergence. Most tests rejected the null hypothesis of neutral evolution for this region and haplotype analysis revealed the presence of two deeply separated clades. Calculation of the time to the most recent common ancestor (TMRCA) for TRIM5 haplotypes yielded estimates ranging between 4 and 7 million years. Overall, these data indicate that long-term balancing selection, an extremely rare process outside MHC genes, has maintained trans-specific polymorphisms in the first intron of TRIM5. Bioinformatic analyses indicated that variants in intron 1 may affect transcription factor-binding sites and, therefore, TRIM5 transcriptional activity. Data herein confirm an extremely complex evolutionary history of TRIM5 genes in primates and open the possibility that regulatory variants in the gene modulate the susceptibility to HIV-1.

Genetic correlates of protection against HIV infection: the ally within.

Repeated exposure to HIV does not necessarily result in infection and HIV infection does not inevitably lead to the development of the AIDS. Multiple immunological and genetic features can confer resistance to HIV acquisition and progression at different steps in viral infection; a full understanding of these mechanisms could result in the development of novel therapeutic and vaccine approaches for HIV infection. In this review, we focus on the genetic mechanisms associated with resistance to HIV infection and to the progression to AIDS.

Different immunologic profiles characterize HIV infection in highly active antiretroviral therapy-treated and antiretroviral-naïve patients with undetectable viraemia. The Master Group.

BACKGROUND: Suppression of human immunodeficiency virus (HIV) replication can be obtained in chronically infected individuals by highly active antiretroviral therapy (HAART) and can also be observed in antiretroviral-naïve patients. The immunological correlates of these two situations were examined. DESIGN AND METHODS: Cross-sectional study involving 32 HIV-infected patients with undetectable HIV plasma viraemia (< 500 copies/ml) and either antiretroviral-naive (n = 14) or undergoing HAART therapy with two nucleoside reverse transcriptase inhibitors (NRTI) plus one (n = 13) or two (n = 5) protease inhibitors (PI). CD4 counts, disease duration, and CDC clinical stage were comparable between the two groups of individuals. Immune parameters (antigen- and mitogen-stimulated proliferation and cytokine production; cytokine mRNA; beta chemokine production; HIV coreceptors mRNA) were analysed in all patients. RESULTS: Results showed immune profiles to be profoundly different in antiretroviral-naive in comparison with HAART-treated patients. Thus: (1) T-cell proliferation to HIV-specific and HIV-unrelated antigens is potent in antiretroviral-naive but suppressed in HAART-treated individuals; (2) interleukin-(IL)2, IL-12 and interferon gamma (IFNgamma) production is robust in naive patients; and (3) a high CCR5/low CXCR4 pattern of HIV coreceptors-specific mRNA is observed in naive but not in HAART-treated patients. In contrast with these observations, no clear differences were detected when beta chemokine production by either peripheral blood mononuclear cells or purified CD8+ T-cells was analysed. Results from HAART-treated patients undergoing therapy with one PI and two NRTI or two PI and two NRTI were in very close agreement. CONCLUSIONS: These data suggest that control over HIV replication can be independently achieved by pharmacological or immunologic means. HAART is associated with weaker HIV-specific and -non-specific immune responses.

A role for mucosal immunity in resistance to HIV infection.

In a recent, thought-provoking novel by Elizabeth McCracken (The Giant's House. Avon Books, New York, 1997), two characters discuss love and its impossibilities. One brashly claims to be "immune to love", explaining the concept to his perplexed interlocutor, "...people become immune to love like they become immune to any disease. Either they had it bad early in life, like chicken pox and that's that; or they keep getting exposed to it in little doses and build up an immunity; or somehow they just don't catch it, something in'em is born resistant. I'm the last type. I'm immune to love and poison ivy". (p. 275) (E. McCracken, The Giant's House. Avon Books, New York, 1997). Substitute the words 'HIV infection' for 'love' and this intriguing metaphor summarizes the state of the art working hypotheses for the phenomenon of resistance to HIV infection in HIV-exposed individuals who, against all odds, do not seroconvert. These hypotheses will be discussed hereafter and particular emphasis will be placed upon a possible role for mucosal immunity in this phenomenon.

Prognostic value of proliferating cell nuclear antigen (PCNA) expression in unresectable hepatocellular carcinoma.

Recent studies indicate that evaluation of cell proliferation in mallignant tumors may have prognostic value, but limited data are available on its expression in hepatocellular carcinoma. We therefore evaluated the prognostic tool of PCNA antigen expression on paraffin-embedded sections of 54 patients with hepatocellular carcinoma and concomitant cirrhosis. According to the number of positive cells, the PCNA expression ranged between 0.8 and 47%, and was divided into two groups (Group A, PCNA less than or equal to 5.5%; Group B, PCNA >5.5%). The two groups of patients were numerically well balanced. Median survival was 16 months for group A and 12 months for group B. According to the Cox multivariate analysis, PCNA expression (P=0.002),TNM stage (P=0.009) and ECOG performance status (P<0.001) significantly correlated with survival. In conclusion, PCNA antigen expression was found to be a significant prognostic faeature in unresectable hepatocellular carcinoma and may be a useful tool for future trials.

Comparison of competitive and non-competitive reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of hepatitis C virus (HCV) RNA.

A competitive reverse transcription (RT)-nested polymerase chain reaction (PCR) assay for HCV RNA was compared with the Roche Amplicor HCV Monitor assay, based on non-competitive, single step RT-PCR. A total of 83 serum samples were tested in parallel by both assays. All samples could be quantified by competitive RT-PCR (cPCR), whereas seven were negative by the non-competitive assay (ncPCR). The HCV RNA titre of the discordant samples assessed by cPCR was significantly lower than that of the remaining 76 (P < 0.001). Absolute HCV RNA titres were higher by cPCR than by ncPCR (P < 0.001), even if the results of the two methods were statistically correlated (P < 0.001). HCV RNA titre tested by cPCR was not significantly different between samples infected with genotype 1 or 2. However, values obtained by ncPCR were higher in samples with genotype 1 (P < 0.001). Furthermore, all seven discordant samples were infected with genotype 2. When both methods were used to measure serial dilutions of standard HCV RNA, we observed a bias to lower measurements with the ncPCR kit. This study shows a good correlation between the results of two PCR-based methods for the quantification HCV RNA. However, the degree of sensitivity and the absolute HCV RNA titre measured may vary according to the assay used.

A DNA hybridization method for typing hepatitis C virus genotype 2c.

A high prevalence of hepatitis C virus (HCV) genotype 2c (22%) was detected in sera from 459 italian patients by core-region amplification and hybridization with specific probes by DNA enzyme immunoassay. Amplified fragments failed to hybridize with 1a, 1b, 2a, 2b and 3a subtype-specific and 4, 5, 6 type-specific oligonucleotides in 105 patients. Hybridization of these samples with type 2 probe, which recognized all the subtypes sequences, showed evidence for genotype 2 distinct from 2a and 2b. Fourteen out of these 105 isolates were cloned and sequenced. The results were consistent with genotyping assay. Nucleotide sequences were partially related to types 2a, 2b, 2d, 2e and 2f (87.0-93.5% of identity). The average nucleotide identity was highest for genotype 2c (95.87%). On the basis of sequence analysis, subtype 2c specific probe was derived. Hybridization efficiency with the newly designed probe was very high and more than 95% (100/105) of type 2 cases were classified as 2c. Evidence of different outcome of therapy inside the same HCV major type account for the need of accurate subtyping. In this study, amplification of the core region followed by hybridization with highly specific probes enabled distinction between HCV subtypes.

Fibrogenesis serum markers in patients with chronic hepatitis C treated with alpha-IFN.

The correlation between therapeutic response and liver fibrogenesis was studied in serum and liver specimens taken from 31 patients treated with alpha-interferon (IFN) (14 sustained responders and 17 non-responders) for chronic hepatitis C. Serum samples, collected before therapy, and at further 6-month intervals over 2 years, were tested for markers of liver neofibrogenesis. Serum N-terminal procollagen III peptide (PIIINP) displayed a significant and persistent decrease (P < 0.05) in sustained responders but not in non-responders; significantly lowered (P < 0.05) mean levels of C-terminal procollagen I peptide (PICP) were transiently observed in both patient groups, apparently as a result of IFN administration. Serum laminin (Lam) levels remained unchanged. One year after the cessation of treatment, liver biopsy re-testing showed an improvement in necro-inflammatory scores only in sustained responders, with the histological fibrosis scores remaining unaltered in both groups. IFN treatment seemed to exert an influence on serum levels of markers of hepatic connective tissue turnover even in patients that did not respond to therapy, while no effect was observed on preexistent liver fibrosis.

HGV/GBV-C infection in patients with acute hepatitis of different etiology and in patients with chronic hepatitis C.

To investigate the prevalence of hepatitis G virus (HGV/GBV-C) in patients with liver disease and to confirm its hypothesized ability to cause liver damage, we studied 130 subjects; 61 had chronic hepatitis C virus infection and 69 had acute hepatitis of either defined etiology (n = 57) or of unknown origin (n = 12). Positivity for HGV/GBV-C RNA was detected in 10 of the 61 subjects with chronic hepatitis C (16.3%) and in 11 of the 57 subjects with acute hepatitis of defined etiology (19%), whereas we failed to detect HGV/ GBV-C viremia in subjects with hepatitis of nonestablished etiology. Patients exhibiting positivity for HGV/GBV-C RNA were found to be comparable to those exhibiting negativity for HGV/GBV-C RNA in terms of both liver function tests and Knodell's score (in liver biopsies); the affect of HGV/GBV-C infection on the biohumoral and histological activity in patients with chronic hepatitis C therefore appears to be minimal or absent. Similar clinical features were observed in patients with acute hepatitis of known etiology whether they were positive or negative for HGV/GBV-C RNA. However, long-term clinical studies are still required to clarify the actual impact of HGV/GBV-C co-infection. In our geographic, i.e., a region or north-east Italy, HGV/GBV-C infection appears to be strictly related to intravenous drug use, and this agent does not seem to be responsible for acute hepatitis of unknown etiology; other etiological agents are probably involved.

Functional analysis of HIV-specific cytotoxic T lymphocytes in antiviral-treated- and-naive patients: a preliminary report.

HIV-specific CTL functions were analyzed in HIV-infected individuals who did or did not receive antiretroviral therapy (ART). Results showed that gp 160 (env)-stimulated perforin- and granzyme-expressing CTL, as well as perforin and granzyme-specific mRNA, were reduced in treated patients whereas TNFalpha was increased in ART-treated compared to naive individuals. Reduction of perforin and granzyme-expressing cells was not secondary to impaired IFNgamma production. A defect of CTL is observed in ART-treated individuals; this defect is not dependent on impaired Th cell function. These results reinforce the need for immunomodulants to successfully approach therapy of HIV infection.