Vascular Endothelial Growth Factor Single Nucleotide Polymorphism +405 G/C (rs2010963) is associated with Levels, Infection Severity, and Amputation among South Indian Diabetic Foot Ulcer Patients.

Background: The regulation of vascular endothelial growth factor (VEGF) by genetic factors in T2DM and DFU still requires thorough investigation. Hence, the present study aimed to investigate the association of VEGF +405 G/C in DFU subjects and correlate it with its circulatory levels, infection severity, and amputation rate. Materials and Methods: This study registered a total of 754 participants of which group I: healthy controls (n = 297), group II: T2DM subjects (n = 242), and group III: DFU subjects (n = 215). Genotyping and levels of rs2010963 were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and ELISA, respectively. Results: Results of the current study showed a clear decline in circulatory VEGF-A levels in DFU subjects. VEGF-A was decreased in DFU subjects with the mutant "CC" genotype. The mutant "CC" of VEGF +405G/C was also found to be more susceptible to ulcer grade (III and IV) and major amputations. Conclusion: VEGF +405G/C SNP is associated with levels, infection severity, and amputation amongst South Indian DFU patients.

Antiretroviral therapy regimen modification rates and associated factors in a cohort of HIV/AIDS patients in Asmara, Eritrea: a 16-year retrospective analysis.

Combined antiretroviral therapy (cART) durability and time to modification are important quality indicators in HIV/AIDs treatment programs. This analysis describes the incidence, patterns, and factors associated with cART modifications in HIV patients enrolled in four treatment centers in Asmara, Eritrea from 2005 to 2021. Retrospective cohort study combining data from 5020 [males, 1943 (38.7%) vs. females, 3077 (61.3%)] patients were utilized. Data on multiple demographic and clinical variables were abstracted from patient's charts and cART program registry. Independent predictors of modification and time to specified events were evaluated using a multi-variable Cox-proportional hazards model and Kaplan-Meier analysis. The median (±IQR) age, CD4+ T-cell count, and proportion of patients with WHO Clinical stage III/IV were 48 (IQR 41-55) years; 160 (IQR 80-271) cells/µL; and 2667 (53.25%), respectively. The cumulative frequency of all cause cART modification was 3223 (64%): 2956 (58.8%) substitutions; 37 (0.7%) switches; and both, 230 (4.5%). Following 241,194 person-months (PMFU) of follow-up, incidence rate of cART substitution and switch were 12.3 (95% CI 11.9-12.8) per 1000 PMFU and 3.9 (95% CI 3.2-4.8) per 10,000 PMFU, respectively. Prominent reasons for cART substitution included toxicity/intolerance, drug-shortage, new drug availability, treatment failure, tuberculosis and pregnancy. The most common adverse event (AEs) associated with cART modification included lipodystrophy, anemia and peripheral neuropathy, among others. In the adjusted multivariate Cox regression model, Organisation (Hospital B: aHR = 1.293, 95% CI 1.162-1.439, p value < 0.001) (Hospital D: aHR = 1.799, 95% CI 1.571-2.060, p value < 0.001); Initial WHO clinical stage (Stage III: aHR = 1.116, 95% CI 1.116-1.220, p value < 0.001); NRTI backbone (D4T-based: aHR = 1.849, 95% CI 1.449-2.360, p value < 0.001) were associated with increased cumulative hazard of treatment modification. Baseline weight (aHR = 0.996, 95% CI 0.993-0.999, p value = 0.013); address within Maekel (aHR = 0.854, 95% CI 0.774-0.942, p value = 0.002); AZT-based backbones (aHR = 0.654, 95% CI 0.515-0.830, p value < 0.001); TDF-based backbones: aHR = 0.068, 95% CI 0.051-0.091, p value < 0.001), NVP-based anchors (aHR = 0.889, 95% CI 0.806-0.980, p value = 0.018) were associated with lower cumulative hazards of attrition. The minimal number of switching suggests inadequate VL testing. However, the large number of toxicity/intolerance and drug-shortage driven substitutions highlight important problems in this setting. Consequently, the need to advocate for both sustainable access to safer ARVs in SSA and improvements in local supply chains is warranted.

Predictive Factors for Submandibular Gland Involvement in Oral Cavity Squamous Cell Carcinoma-a Prospective Study from a Tertiary Cancer Center.

The oncological necessity of submandibular gland removal during neck dissection for oral cavity squamous cell carcinoma surgery has remained controversial. This study was aimed to determine the rate of SMG involvement and assess the feasibility of submandibular gland (SMG) preservation. We present a prospective study conducted at a tertiary cancer center from June 2017 to May 2019. All patients of oral squamous cell carcinoma who underwent primary surgery with neck dissection were included and analyzed for incidence and predictive factors for incidence of level IB nodal and SMG involvement as per CAP guidelines. A total of 60 patients were inducted in the study, wherein 63 neck dissections were performed including bilateral dissection in three cases. There was involvement of SMG in 6 patients with two cases each in floor of mouth cancer, gingivo-buccal, and alveolar lesions. The SMG was involved by direct contiguous spread from the primary lesion in two cases, extra-capsular extension from level IB lymph nodes in one and by both mode of spread in three glands. Perineural invasion was seen in 83.33% (n = 5) patients with SMG involvement (p- < 0.001), while 66.67% (4/6) patients had lympho vascular invasion (p-0.006) and all the cases with SMG involvement had extra-capsular extension (p < 0.001), suggesting PNI, LVI, and ECE as the strongest predictors of SMG involvement. This study demonstrates that oral cavity squamous cell carcinoma has low potential to metastasize to the SMG; however, high-risk factors include primary tumor site in floor of mouth or tongue, heavy level IB nodal burden, presence of LVI, PNI, and ECE. In the absence of these high-risk factors, SMG preservation with complete nodal clearance in level IB is a promising technique for reducing future complications.

Can Preoperative Ultrasonography and MRI Replace Sentinel Lymph Node Biopsy in Management of Axilla in Early Breast Cancer-a Prospective Study from a Tertiary Cancer Center.

Although SLNB is a less invasive procedure in detecting axillary lymph node metastases(ALNM) in early breast cancer; still, it carries some complications like lymphedema and in addition, performing SLNB requires surgical skills, technical knowledge, presence of facility like preoperative sentinel lymphoscintigraphy, and availability of hand-held gamma probe for intraoperative assessment. We calculated the relative diagnostic strength of preoperative axillary USG and MRI and compared with of SLNB for detection of ALNM in early breast cancer and assessed whether MRI and USG could accurately predict axillary LN status, potentially replacing SLNB. We evaluated 40 cases of clinically node-negative early breast cancer with preoperative axillary USG and MRI and subsequently subjected to SLNB. The sensitivity, specificity, PPV, NPV, and accuracy of axillary USG were 62.5%, 96.88%, 88.33%, 91.18%, and 90% respectively (p value < 0.001). The sensitivity, specificity, PPV, NPV, and accuracy of MRI in detection of ALNM were 75%, 93.75%, 75%, 93.75%, and 90% (p value < 0.001). The sensitivity, specificity, PPV, NPV, and accuracy of combined USG and MRI in detection of ALNM were 87.5%,90.63%, 70%, 96.67%, and 90% respectively (p value < 0.001), which are comparable to previous study series. The diagnostic performance of combined approach of axillary USG and MRI is promising, as the NPV of combined USG and MRI is approaching the NPV of the SLNB in detecting ALNM. Based on above findings, if axillary LNs are found nonsuspicious in preoperative axillary USG and MRI, further axillary dissection may be avoided, and if found suspicious, then ALND may be directly proceeded avoiding SLNB in between.

Decreased TNF family receptor expression on B-cells is associated with reduced humoral responses to Streptococcus pneumoniae infections in young children.

An underdeveloped or impaired immune response in young children is associated with increased susceptibility to Streptococcus pneumonia (Spn) infections. We determined serum antibody titers against 3 Spn vaccine candidate proteins and vaccine serotype polysaccharide antigens in a group of Spn infection prone 9-18months old and found lower IgG antibody titers to all tested antigens compared to age-matched non-infection-prone children. We also found the children had significantly reduced percentages of total memory B-cells, switched memory B-cells and plasma cells. We sought a mechanistic explanation for that result by examination of TNF family receptors (TNFRs) TACI, BCMA, and BAFFR receptor expression on B-cells and found significantly lower BAFFR and TACI expression; significantly lower proliferation of B-cells stimulated with exogenous BAFF; and diminished expression of co-stimulatory receptors B7-1 and B7-2 among infection prone vs. non-prone children. We conclude that lower expression of TNFRs, lower proliferation of B-cells in response to BAFF and lower expression of B7-1 and B7-2 by B-cells may contribute to reduced antibody responses to Spn and consequent infection proneness in young children.

Reduced T-Helper 17 Responses to Streptococcus pneumoniae in Infection-Prone Children Can Be Rescued by Addition of Innate Cytokines.

Background: T-helper (Th) 17 cells are important in the control of Streptococcus pneumoniae. We sought to understand the mechanism of failure of Th17 immunity resulting in S. pneumoniae infections in children <2 years old. Methods: Peripheral blood mononuclear cells (PBMCs) from infection-prone (IP) and non-IP (NIP) children 9-18 months old were examined for their responses to heat-killed S. Pneumoniae, using flow cytometry, reverse-transcription polymerase chain reaction, and enzyme-linked immunoassay. We measured cytokine production, proliferation, and differentiation of Th17 cells and the expression of transcription factors in response to S. pneumoniae. Results: PBMCs of IP children stimulated with heat-killed S. pneumoniae had significantly reduced percentages of CD4+ Th1 (interleukin2, tumor necrosis factor α) and Th17 (interleukin 17A) cells compared with NIP children. Addition of exogenous Th17-promoting cytokines (interleukin 6, 1ß, and 23 and transforming growth factor ß) restored CD4+ Th17 cell function in cells from IP children to levels measured in NIP children. Conclusions: Reduced Th17 responses to S. pneumoniae in PBMCs of IP children can be rescued by addition of Th17-promoting cytokines.

Developing a vaccine to prevent otitis media caused by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a predominant organism of the upper respiratory nasopharyngeal microbiota. Its disease spectrum includes otitis media, sinusitis, non-bacteremic pneumonia and invasive infections. Protein-based vaccines to prevent NTHi infections are needed to alleviate these infections in children and vulnerable populations such as the elderly and those with chronic obstructive pulmonary disease (COPD). One NTHi protein is included in a pneumococcal conjugate vaccine and has been shown to provide efficacy. Our lab has been interested in understanding the immunogenicity of NTHi vaccine candidates P6, protein D and OMP26 for preventing acute otitis media in young children. We expect that continued investigation and progress in the development of an efficacious protein based vaccine against NTHi infections is achievable in the near future.

Functional Immune Cell Differences Associated With Low Vaccine Responses in Infants.

BACKGROUND: We sought to understand why some children respond poorly to vaccinations in the first year of life. METHODS: A total of 499 children (6-36 months old) provided serum and peripheral blood mononuclear cell samples after their primary and booster vaccination. Vaccine antigen-specific antibody levels were analyzed with enzyme-linked immunosorbent assay, and frequency of memory B cells, functional T-cell responses, and antigen-presenting cell responses were assessed in peripheral blood mononuclear cell samples with flow cytometric analysis. RESULTS: Eleven percent of children were low vaccine responders, defined a priori as those with subprotective immunoglobulin G antibody levels to ≥66% of vaccines tested. Low vaccine responders generated fewer memory B cells, had reduced activation by CD4(+) and CD8(+) T cells on polyclonal stimulation, and displayed lower major histocompatibility complex II expression by antigen-presenting cells. CONCLUSIONS: We conclude that subprotective vaccine responses in infants are associated with a distinct immunologic profile.

A comparative study on serum lipoprotein (a) and lipid profile between rheumatoid arthritis patients and normal subjects.

BACKGROUND: Rheumatoid arthritis is a chronic and systemic inflammatory disorder, in which Lipoprotein (a) [Lp (a)] increases plaque formation and thus promotes atherosclerosis. Coronary artery disease is one of the co-morbidity in rheumatoid arthritis patients. AIM: The aim of this study is to evaluate Lp (a) as a cardiovascular risk factor in patients with rheumatoid arthritis. This was a comparative study in which Lp (a) and lipid profile were compared in rheumatoid arthritis patients and controls. MATERIALS AND METHODS: The study included 30 sero-positive rheumatoid arthritis patients and 30 normal healthy subjects with an age and sex matched group of 25-80 years. Statistical analysis was performed using SPSS version 17. RESULTS: Serum Lp (a) concentration was significantly increased (P < 0.001) in rheumatoid arthritis patients compared with controls. Serum high-density lipoprotein-cholesterol was significantly lowered (P < 0.05) in patients as compared to controls. There was no significant difference in serum total cholesterol, triglycerides, and very low density lipoprotein-cholesterol between patients and controls. CONCLUSION: The findings indicate that the patients with rheumatoid arthritis are at high risk of developing cardiovascular disease in future due to the increased level of Lp (a). In addition to conventional lipid profile, estimation of Lp (a) can prove to be a valuable tool in risk assessment of population in general and management of disease in particular.

Immune responses in neonates.

Neonates have little immunological memory and a developing immune system, which increases their vulnerability to infectious agents. Recent advances in the understanding of neonatal immunity indicate that both innate and adaptive responses are dependent on precursor frequency of lymphocytes, antigenic dose and mode of exposure. Studies in neonatal mouse models and human umbilical cord blood cells demonstrate the capability of neonatal immune cells to produce immune responses similar to adults in some aspects but not others. This review focuses mainly on the developmental and functional mechanisms of the human neonatal immune system. In particular, the mechanism of innate and adaptive immunity and the role of neutrophils, antigen presenting cells, differences in subclasses of T lymphocytes (Th1, Th2, Tregs) and B cells are discussed. In addition, we have included the recent developments in the neonatal mouse immune system. Understanding neonatal immunity is essential to development of therapeutic vaccines to combat newly emerging infectious agents.

Comparison of antibody and T-cell responses elicited by licensed inactivated- and live-attenuated influenza vaccines against H3N2 hemagglutinin.

T cells are being increasingly recognized as a significant component of influenza-specific immune responses in humans. Although an inactivated- and a live-attenuated influenza vaccine are now licensed for use in humans, their comparative ability to elicit T-cell responses against influenza is not well understood. Using the rapidly evolving H3N2 hemagglutinin (HA) as an antigenic model, we compared immune responses elicited by the trivalent inactivated influenza vaccine (TIV) and the live-attenuated influenza vaccine (LAIV) in a cohort of healthy adults 18-49 years of age. TIV elicited higher geometrical mean antibody titers than LAIV, whereas, LAIV elicited superior T-cell responses. Importantly, LAIV elicited higher magnitude T-cell responses toward the rapidly drifting variant region of HA that is prone to escape from antibody responses. These results have important implications for the deployment of influenza vaccines in years of antigenic mismatch and shift.

Selection of peptide ligands for human placental transcytosis systems using in vitro phage display.

Fetal pharmacotherapy generally relies on nonspecific biodistribution of therapeutic agents to the unborn child following drug administration into the maternal circulation system. Physiologically, transfer of polar, high-molecular weight solutes across the placenta is facilitated by a specialized, vesicular transport mechanism termed transcytosis. To develop biotechnology-based drugs such as proteins, DNA, and siRNA as clinically effective therapeutics, transcytosis systems have been evaluated as a promising strategy to augment drug transfer across endothelial and epithelial barriers. Screening of random peptide libraries using phage display is a powerful technology to identify peptide sequences with high affinity for surface proteins on desired target cells. Here, we describe assembly of a diverse, cyclic heptapeptide library on the icosahedral T7 bacteriophage platform. This phage-displayed library of random peptides was used for functional in vitro screens across BeWo cell monolayers to identify peptide ligands that facilitate placental transcytosis of viral particles across this cell culture model of the human trophoblast barrier.

Pandemic and seasonal H1N1 influenza hemagglutinin-specific T cell responses elicited by seasonal influenza vaccination.

Understanding whether seasonal influenza vaccines can elicit antibody and T cell responses against the 2009 pandemic H1N1 strain is important. We compared T cell and antibody responses elicited by trivalent inactivated influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV) in healthy adults. Both vaccines boosted pre-existing T cells to the seasonal and pandemic hemagglutinin (HA) but responses were significantly greater following immunization with LAIV. Antibody titers were significantly boosted only by TIV. The relationship between antibody and T cell responses and the effect of the magnitude of pre-existing immunity on vaccine-induced responses were also evaluated. Cross reactive T cell responses to the pandemic H1N1 HA existed among the cohort before the circulation of the virus to varying degrees and these responses were boosted by seasonal vaccination.

Age-related changes in magnitude and diversity of cross-reactive CD4+ T-cell responses to the novel pandemic H1N1 influenza hemagglutinin.

Accumulating evidence suggests that T-cell responses play a significant role in controlling influenza disease. Although humoral responses to the major antigenic component hemagglutinin (HA) have been studied in considerable detail, our understanding of the cellular responses against this antigen is limited. Here, we systematically characterized the magnitude and diversity of immunity to pandemic H1N1 HA and its relationship to seasonal H1N1 HA responses in a cohort of healthy nonimmunized adults. We observed considerable diversity in the magnitude of crossreactive pandemic CD4+ T-cell responses among the subjects, with a subset of the individuals demonstrating higher magnitude T-cell responses against the pandemic antigen compared with the seasonal antigen. Importantly, the data suggest that age-related changes in CD4+ T-cell responses preferentially segregate to the antigenically drifting globular region of HA, more so than the conserved region involved in membrane fusion. These results have important ramifications for our understanding of influenza immunity in humans and development of vaccine strategies against this important pathogen.

Polyvalent inhibitors of anthrax toxin that target host receptors.

Resistance of pathogens to antimicrobial therapeutics has become a widespread problem. Resistance can emerge naturally, but it can also be engineered intentionally, which is an important consideration in designing therapeutics for bioterrorism agents. Blocking host receptors used by pathogens represents a powerful strategy to overcome this problem, because extensive alterations to the pathogen may be required to enable it to switch to a new receptor that can still support pathogenesis. Here, we demonstrate a facile method for producing potent receptor-directed antitoxins. We used phage display to identify a peptide that binds both anthrax-toxin receptors and attached this peptide to a synthetic scaffold. Polyvalency increased the potency of these peptides by >50,000-fold in vitro and enabled the neutralization of anthrax toxin in vivo. This work demonstrates a receptor-directed anthrax-toxin inhibitor and represents a promising strategy to combat a variety of viral and bacterial diseases.

Statistical pattern matching facilitates the design of polyvalent inhibitors of anthrax and cholera toxins.

Numerous biological processes involve the recognition of a specific pattern of binding sites on a target protein or surface. Although ligands displayed by disordered scaffolds form stochastic rather than specific patterns, theoretical models predict that recognition will occur between patterns that are characterized by similar or "matched" statistics. Endowing synthetic biomimetic structures with statistical pattern matching capabilities may improve the specificity of sensors and resolution of separation processes. We demonstrate that statistical pattern matching enhances the potency of polyvalent therapeutics. We functionalized liposomes with an inhibitory peptide at different densities and observed a transition in potency at an interpeptide separation that matches the distance between ligand-binding sites on the heptameric component of anthrax toxin. Pattern-matched polyvalent liposomes inhibited anthrax toxin in vitro at concentrations four orders of magnitude lower than the corresponding monovalent peptide, and neutralized this toxin in vivo. Statistical pattern matching also enhanced the potency of polyvalent inhibitors of cholera toxin. This facile strategy should be broadly applicable to the detection and neutralization of toxins and pathogens.