Evolution of COVID-19 infection in Punjab; trends during five waves of infection in the province of Punjab.

BACKGROUND: Pakistan witnessed five waves of COVID-19 infections during the pandemic. Punjab, the largest province of Pakistan, remained the epicentre due to a high infection rate. Administrative data for five waves of the pandemic was analyzed to determine the rate of infections and the significance of pharmacological and non-pharmacological interventions on the severity and duration of infection. METHODOLOGY: COVID-19 data from March 2020 to May 2023 was obtained from the Provincial Public Health Reference Laboratory (PPHRL), Punjab AIDS Control Program, Lahore. The data included samples from index cases, contacts, and recovered patients. A total of 36,252,48 cases were screened for COVID-19, and 90,923 (2.50%) were detected positive by RT-PCR, accounting for 5.69% of the cases reported positive throughout the country. RESULTS: Among the positive cases, 50.86% (n = 46,244) cases were new cases (registered for the first time), 40.41% (n = 36751) were the contact cases traced from the newly identified cases and 8.62% (n = 7842) repeated cases. The positivity rates among index cases were reported to be 2.37%, 2.34%, 4.61%, 2.09%, and 1.19%, respectively, for the five respective COVID-19 pandemic waves. Distribution by gender indicated that 64% of males and 35% of females were infected during the pandemic. The age factor demonstrated the most susceptibility to infection in women aged 19-29 years, whereas most males between the ages of 29-39 had an infection. Susceptibility to COVID-19 infection was observed to be equally likely between males and females; however, clinical outcomes indicated that infections in males were more severe and often resulted in fatalities as compared to those in females. This trend was also reflected in the viral titer as measured by the Ct values, where 40% of males had Ct values < 25 (an indicator of high viral titers) compared to 30% of females with Ct values < 25. CONCLUSION: Overall, our data indicated that infection rates remained stable throughout the pandemic except for 3rd wave, which showed a higher incidence of infection rate of 4%. Additionally, data showed a positive impact of masking, social distancing, and immunization, as indicated by the shorter window of high infection rates.

Strain features of pneumococcal isolates in the pre- and post-PCV10 era in Pakistan.

Pakistan is amongst the four countries with the highest number of pneumococcal deaths. While the PCV10 vaccine was introduced in Pakistan in October 2012, data regarding the impact of the vaccine on the population dynamics of Streptococcus pneumoniae in Pakistan remain obscure. Using whole genome sequencing of 190 isolates (nasopharyngeal carriage=75, disease=113, unknown sites=2) collected between 2002 and 2020, this study presents characteristics of pneumococcal strains in Pakistan in the pre- and post-vaccine era. The isolates were characterized on the basis of serotype distribution, genetic lineages (or Global Pneumococcal Sequence Cluster, GPSC) and antibiotic resistance. A high level of diversity in serotype and genetic lineages of pneumococci was observed in Pakistan. Among 190 isolates, we identified 54 serotypes, 67 GPSCs and 116 sequence types (STs) including 23 new STs. The most prevalent GPSCs and their associated serotypes in nasopharyngeal carriage were GPSC54 (expressing serotype 9V), GPSC5 (15A and 7B, and serogroup 24), GPSC25 (15B/15C), GPSC67 (18C) and GPSC376 (6A and 6D). Similarly, among 113 disease-causing isolates, the most prevalent GPSC/serotype combinations were GPSC2 (serotype 1), GPSC10 (serotypes 14, 10A, 19A and 19F), GPSC43 (serotypes 13, 11A, 23B, 35A and 9V), GPSC67 (serotypes 18A and 18C) and GPSC642 (serotype 11A). Of the 190 isolates, the highest levels of resistance were observed against penicillin (58.9 %, n=122), erythromycin (29.5 %, n=56), clindamycin (13.2 %, n=25), co-trimoxazole (94.2 %, n=179) and tetracycline/doxycycline (53.2 %, n=101). A higher proportion of disease-causing isolates were multidrug resistant as compared to carriage isolates (54 % vs 25 %). Our data suggest limited coverage of PCV10 in nasopharyngeal (21.6 %, 16/74) as well as disease-causing (38.1 %, 16/42) isolates among children ≤5 years old; however, higher valent vaccine PCV13 would increase the coverage rates to 33.8 % in nasopharyngeal and 54.8 % in disease-causing isolates, whereas PCV24/25 would offer the highest coverage rates. Owing to the diversity of serotypes observed during the post-vaccine period, the suggested inclusion of serotype in future vaccine formulations will require investigations with larger data sets with an extended temporal window. This article contains data hosted by Microreact.

Regional and National Trends in Consumption of Antimicrobials in Pakistan; Pre and Post-COVID (2019-2021).

BACKGROUND: Efforts to combat antimicrobial resistance, a growing public health problem in Pakistan, have been hampered by the lack of high-quality national and provincial-level antimicrobial consumption data. The singular objective of this retrospective study was to measure antimicrobial consumption over 3 years between 2019 and 2021. METHODS: The study was designed to estimate antimicrobial consumption at National and Regional levels. Antimicrobial consumption data was collected by IQVIA covering 110 districts of Pakistan in which 88% of sales are census (accurate sales collected directly from distributors), whereas 12% of sales (sales of 300 pharmacies) are projected on the national level. To determine the usage for 3 consecutive years, the consumption of antibiotics was calculated as defined daily doses (DDD) of antibiotics per 1000 inhabitants per day (DID). RESULTS: The results of our study demonstrated a steep increase in the consumption of antimicrobials from 2019 to 2021. An increase in consumption of most classes of antibiotics was observed both nationally and Regionally. Quinolones, penicillins (co-amoxiclav), macrolides, and third-generation cephalosporins remained the most frequently used antibiotics nationally. A 40% increase in intravenous use of antimicrobials was observed between 2019 and 2021 at the national level. Moxifloxacin, Levofloxacin, Ciprofloxacin, and linezolid were the most commonly used intravenous antibiotics. Region 7 (Peshawar) demonstrated the highest consumption, followed by Region 1 (Karachi) and Region 6 (Faisalabad). Among the most commonly used antibiotics, the use of third-generation cephalosporin (cefixime), quinolones, penicillins (amoxicillin + clavulanic acid), and macrolides (azithromycin) was most noticeable in all regions, particularly in those with the higher consumption of antibiotics. CONCLUSIONS: Although the increase in consumption of all antibiotics is concerning, the steep increase in the use of watch and reserve category antibiotics during the study period calls for immediate actions to limit and regulate their usage.

Spontaneous NETosis in diabetes: A role of hyperglycemia mediated ROS and autophagy.

Type 2-diabetes, particularly poorly controlled diabetes, is a risk factor for several infections such as lower respiratory tract and skin infections. Hyperglycemia, a characteristic downstream effect of poorly controlled diabetes, has been shown to impair the function of immune cells, in particular neutrophils. Several studies have demonstrated that hyperglycemia-mediated priming of NADPH oxidase results in subsequent elevated levels of reactive oxygen species (ROS). In healthy neutrophils, ROS plays an important role in pathogen killing by phagocytosis and by induction of Neutrophil Extracellular Traps (NETs). Given the key role of ROS in autophagy, phagocytosis and NETosis, the relationship between these pathways and the role of diabetes in the modulation of these pathways has not been explored previously. Therefore, our study aimed to understand the relationship between autophagy, phagocytosis and NETosis in diabetes. We hypothesized that hyperglycemia-associated oxidative stress alters the balance between phagocytosis and NETosis by modulating autophagy. Using whole blood samples from individuals with and without type 2-diabetes (in the presence and absence of hyperglycemia), we demonstrated that (i) hyperglycemia results in elevated levels of ROS in neutrophils from those with diabetes, (ii) elevated levels of ROS increase LCIII (a marker for autophagy) and downstream NETosis. (iii) Diabetes was also found to be associated with low levels of phagocytosis and phagocytic killing of S. pneumoniae. (iv) Blocking either NADPH oxidase or cellular pathways upstream of autophagy led to a significant reduction in NETosis. This study is the first to demonstrate the role of ROS in altering NETosis and phagocytosis by modulating autophagy in type 2-diabetes. GRAPHICAL ABSTRACT.

Identification of Multiple Variant Extensively Drug-Resistant Typhoid Infections across Pakistan.

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening bacterial infection. Recently, an outbreak of a new sublineage of extensively drug resistant (XDR) S. Typhi emerged in Pakistan in the province of Sindh. This sublineage had both a composite multidrug resistance transposon integrated on the chromosome and an acquired IncY plasmid carrying the extended spectrum beta-lactamase, blaCTX-M-15, which conferred resistance to third-generation cephalosporins. We observed previously that XDR typhoid had spread beyond the originating southern Sindh Province. Thus, we sought to determine the genetic diversity of 58 ceftriaxone-resistant S. Typhi clinical isolates by whole genome sequencing collected across Pakistan from November 2018 to December 2020 to provide insights into the molecular epidemiology of the evolving outbreak. We identify multiple novel genomic integrations of the extended spectrum beta-lactamase gene into the chromosome in S. Typhi, revealing the existence of various XDR typhoid variants circulating in the country. Notably, the integration of the IncY plasmid bearing antibiotic resistance genes may allow for subsequent plasmid acquisition by these variants, potentially leading to further plasmid-borne multidrug resistance. Our results can inform containment initiatives, help track associated outcomes and international spread, and help determine how widespread the risk is.

Raman spectroscopy and machine learning-based optical probe for tuberculosis diagnosis via sputum.

Tuberculosis (TB) is a contagious disease that causes 1.5 million deaths per year globally. Early diagnosis of TB patients is critical to control its spread. However, standard TB diagnostic tests such as sputum culture take days to weeks to produce results. Here, we demonstrate a quick, portable, easy-to-use, and non-invasive optical sensor based on sputum samples for TB detection. The probe uses Raman spectroscopy to detect TB in a patient's sputum supernatant. We deploy a machine-learning algorithm, principal component analysis (PCA), on the acquired Raman data to enhance the detection sensitivity and specificity. On testing 112 potential TB patients, our results show that the developed probe's accuracy is 100% for true-positive and 93.4% for true-negative. Moreover, the probe correctly identifies patients on TB medication. We anticipate that our work will lead to a viable and rapid TB diagnostic platform.

Emergence of a multidrug-resistant and virulent Streptococcus pneumoniae lineage mediates serotype replacement after PCV13: an international whole-genome sequencing study.

BACKGROUND: Serotype 24F is one of the emerging pneumococcal serotypes after the introduction of pneumococcal conjugate vaccine (PCV). We aimed to identify lineages driving the increase of serotype 24F in France and place these findings into a global context. METHODS: Whole-genome sequencing was performed on a collection of serotype 24F pneumococci from asymptomatic colonisation (n=229) and invasive disease (n=190) isolates among individuals younger than 18 years in France, from 2003 to 2018. To provide a global context, we included an additional collection of 24F isolates in the Global Pneumococcal Sequencing (GPS) project database for analysis. A Global Pneumococcal Sequence Cluster (GPSC) and a clonal complex (CC) were assigned to each genome. Phylogenetic, evolutionary, and spatiotemporal analysis were conducted using the same 24F collection and supplemented with a global collection of genomes belonging to the lineage of interest from the GPS project database (n=25 590). FINDINGS: Serotype 24F was identified in numerous countries mainly due to the clonal spread of three lineages: GPSC10 (CC230), GPSC16 (CC156), and GPSC206 (CC7701). GPSC10 was the only multidrug-resistant lineage. GPSC10 drove the increase in 24F in France and had high invasive disease potential. The international dataset of GPSC10 (n=888) revealed that this lineage expressed 16 other serotypes, with only six included in 13-valent PCV (PCV13). All serotype 24F isolates were clustered in a single clade within the GPSC10 phylogeny and long-range transmissions were detected from Europe to other continents. Spatiotemporal analysis showed GPSC10-24F took 3-5 years to spread across France and a rapid change of serotype composition from PCV13 serotype 19A to 24F during the introduction of PCV13 was observed in neighbouring country Spain. INTERPRETATION: Our work reveals that GPSC10 alone is a challenge for serotype-based vaccine strategy. More systematic investigation to identify lineages like GPSC10 will better inform and improve next-generation preventive strategies against pneumococcal diseases. FUNDING: Bill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

Host biomarkers for monitoring therapeutic response in extrapulmonary tuberculosis.

PURPOSE: The aim of this study was to explore the utility of inflammatory biomarkers in the peripheral blood to predict response to treatment in extrapulmonary tuberculosis (EPTB). METHODS: A Luminex xMAP-based multiplex immunoassay was used to measure 40 inflammatory biomarkers in un-stimulated plasma of 91 EPTB patients (48 lymphadenitis, and 43 pleuritis) before and at 2 and 6 months of treatment. RESULTS: Overall a significant change was observed in 28 inflammatory biomarkers with treatment in EPTB patients. However, MIG/CXCL9, IP-10/CXCL10, and CCL23 decreased in all patients' groups with successful treatment at both time points. At 2 months, 29/64 (45%) patients responded partially while 35/64 (55%) showed complete regress. Among good responders, a higher number of biomarkers (16/40) reduced significantly as compared to partial responders (1/40). Almost half (14/29) of partial responders required longer treatment than 6 months to achieve satisfactory response. The levels of MIG, IP-10, MIF, CCL22 and CCL23 reduced significantly among 80, 74, 60, 71, 51% good responders, as compared to 52, 52, 52, 59, 52% partial responders, respectively. A biosignature, defined by a significant decrease in any one of these five biomarkers, corresponded with satisfactory response to treatment in 97% patients at 2 month and 99% patients at 6 months of treatment. CONCLUSION: Change in inflammatory biomarkers correlates with treatment success. A five biomarker biosignature (MIG, IP-10, MIF, CCL22 and CCL23) could be used as an indicator of treatment success.

Trends in antimicrobial resistance amongst pathogens isolated from blood and cerebrospinal fluid cultures in Pakistan (2011-2015): A retrospective cross-sectional study.

While antimicrobial resistance (AMR) continues to be a major public health problem in Pakistan, data regarding trends of resistance among pathogenic bacteria remains scarce, with few studies presenting long-term trends in AMR. This study was therefore designed to analyze long-term AMR trends at a national level in Pakistan. We report here results of a comprehensive analysis of resistance, among pathogens isolated from blood and cerebrospinal fluid (CSF), between 2011 and 2015. Susceptibility data was obtained from a local laboratory with collection points all across Pakistan (Chughtai Laboratory). Resistance proportions to most commonly used antimicrobials were calculated for each pathogen over a period of five years. While Acinetobacter species demonstrated highest resistance rates to all tested antimicrobials, a sharp increase in carbapenem resistance was the most noticeable (50%-95%) between 2011-2015. Our results also highlight the presence of third and fourth generation cephalosporins resistance in Salmonella enterica serovar Typhi in Pakistan. Interestingly, where rise in AMR was being observed in some major invasive pathogens, decreasing resistance trends were observed in Staphylococcus aureus, against commonly used antimicrobials. Overall pathogens isolated from blood and CSF between 2011-2015, showed an increase in resistance towards commonly used antimicrobials.

An evaluation of lipid profile and pro-inflammatory cytokines as determinants of cardiovascular disease in those with diabetes: a study on a Mexican American cohort.

Sedentary life styles coupled with high-calorie diets and unhealthy social habits such as smoking, have put an ever-increasing number of people at risk of cardiovascular disorders (CVD), worldwide. A concomitant increase in the prevalence of type 2-diabetes (hyperglycemia), a risk factor for CVD, has further contributed towards escalating CVD-related mortalities. The increase in number of cases of type 2-diabetes underscores the importance of early diagnosis of cardiovascular disease in those with diabetes. In this work, we have evaluated the sensitivity and specificity of dyslipidemia and proinflammatory cytokines to be used as biomarkers for predicting the risk of CVD in those with diabetes. We hypothesize that interplay between dyslipidemia and diabetes-induced low-grade inflammation in those with type 2-diabetes increases the risk of CVD. A total of 215 participants were randomly recruited from the Cameron County Hispanic Cohort (CCHC). Of these, 99% were Mexican Americans living on Texas-Mexico border. Levels of cytokines, adipokines and lipid profile were measured. Cardiovascular disease (CVD) for this study was defined as prior diagnosis of heart attack, angina and stroke, while diabetes was defined by fasting blood glucose (FBG) of > 100 mg/dL and HbA1c of > 6.5, in accordance with American Diabetes Association (ADA) guidelines. Depending on type and distribution of data, various statistical tests were performed. Our results demonstrated higher rates of heart attack (14% vs 11.8%) and stroke (19.8% vs 10%) in those with diabetes as compared to non-diabetes. The odds of having a heart attack were eight times higher in the presence of elevated triglycerides and pro-inflammatory markers (TNFα and IL6) as compared to presence of pro-inflammatory markers only. The odds for heart attack among those with diabetes, increased by 20 fold in presence of high levels of triglycerides, TNFα, and IL6 when coupled with low levels of high-density lipid cholesterol (HDL-C). Lastly, our analysis showed that poorly controlled diabetes, characterized by HbA1c values of > 6.5 increases the odds of stroke by more than three fold. The study quantifies the role of lipid profile and pro-inflammatory markers in combination with standard risk factors towards predicting the risk of CVD in those with type 2-diabetes. The findings from the study can be directly translated for use in early diagnosis of heart disease and guiding interventions leading to a reduction in CVD-associated mortality in those with type 2-diabetes.

An Engineered Microvirin Variant with Identical Structural Domains Potently Inhibits Human Immunodeficiency Virus and Hepatitis C Virus Cellular Entry.

Microvirin (MVN) is one of the human immunodeficiency virus (HIV-1) entry inhibitor lectins, which consists of two structural domains sharing 35% sequence identity and contrary to many other antiviral lectins, it exists as a monomer. In this study, we engineered an MVN variant, LUMS1, consisting of two domains with 100% sequence identity, thereby reducing the chemical heterogeneity, which is a major factor in eliciting immunogenicity. We determined carbohydrate binding of LUMS1 through NMR chemical shift perturbation and tested its anti-HIV activity in single-round infectivity assay and its anti-hepatitis C virus (HCV) activity in three different assays including HCVcc, HCVpp, and replicon assays. We further investigated the effect of LUMS1 on the activation of T helper (Th) and B cells through flow cytometry. LUMS1 showed binding to (1-2)mannobiose, the minimum glycan epitope of MVN, potently inhibited HIV-1 and HCV with EC50 of 37.2 and 45.3 nM, respectively, and showed negligible cytotoxicity with CC50 > 10 µM against PBMCs, Huh-7.5 and HepG2 cells, and 4.9 µM against TZM-bl cells. LUMS1 did not activate Th cells, and its stimulatory effect on B cells was markedly less as compared to MVN. Together, with these effects, LUMS1 represents a potential candidate for the development of antiviral therapies.

PspA facilitates evasion of pneumococci from bactericidal activity of neutrophil extracellular traps (NETs).

Pneumococcal strains are variably resistant to killing by neutrophil extracellular traps (NETs). We hypothesize that this variability in resistance is due to heterogeneity in pneumococcal surface protein A (PspA), a structurally diverse virulence factor of Streptococcus pneumoniae. Pneumococcal strains showed variability in induction of NETs and in susceptibility to killing by NETs. The variability in susceptibility to NETs-mediated killing of pneumococcal strains is attributed to PspA, as strains lacking the surface expression of PspA were significantly more sensitive to NETs-mediated killing compared to the wild-type strains. Using pspA switch mutants we were further able to demonstrate that NETs induction and killing by NETs is a function of PspA as mutants with switch PspA demonstrated donor phenotype. Antibody to PspA alone showed an increase in induction of NETs, and NETs thus generated were able to trap and kill pneumococci. Pneumococci opsonized with antibody to PspA showed increase adherence to NETs but a decrease susceptibility to killing by NETs. In conclusion we demonstrate a novel role for pneumococcal PspA in resisting NETs mediated killing and allowing the bacteria to escape containment by blocking binding of pneumococci to NETs.

Estimation of hepatitis C prevalence in the Punjab province of Pakistan: A retrospective study on general population.

BACKGROUND: Hepatitis C virus (HCV) infections are amongst the leading public health concerns in Pakistan with a high disease burden. Despite the availability of effective antiviral treatments in the country the disease burden in general population has not lowered. This could be attributed to the asymptomatic nature of this infection that results in lack of diagnosis until the late symptomatic stage. To better estimate and map HCV infections in the country a population-based analysis is necessary for an effective control of the infection. METHODS: Serologic samples of ~66,000 participants from all major cities of the Punjab province were tested for anti-HCV antibodies. The antibody-based seroprevalence was associated with socio-demographic variables including geographical region, age, gender and sex, and occupation. RESULTS: Overall serological response to HCV surface antigens was observed in over 17% of the population. Two of the districts were identified with significantly high prevalence in general population. Analysis by occupation showed significantly high prevalence in farmers (over 40%) followed by jobless and retired individuals, laborers and transporters. A significant difference in seroprevalence was observed in different age groups amongst sex and genders (male, female and transgender) with highest response in individuals of over 40 years of age. Moreover, most of the tested IDUs showed positive response for anti-HCV antibody. CONCLUSION: This study represents a retrospective analysis of HCV infections in general population of the most populated province of Pakistan to identify socio-demographic groups at higher risk. Two geographical regions, Faisalabad and Okara districts, and an occupational group, farmers, were identified with significantly high HCV seroprevalence. These socio-demographic groups are the potential focused groups for follow-up studies on factors contributing to the high HCV prevalence in these groups towards orchestrating effective prevention, control and treatment.

Evolution of efficacious pangenotypic hepatitis C virus therapies.

Hepatitis C compromises the quality of life of more than 350 million individuals worldwide. Over the last decade, therapeutic regimens for treating hepatitis C virus (HCV) infections have undergone rapid advancements. Initially, structure-based drug design was used to develop molecules that inhibit viral enzymes. Subsequently, establishment of cell-based replicon systems enabled investigations into various stages of HCV life cycle including its entry, replication, translation, and assembly, as well as role of host proteins. Collectively, these approaches have facilitated identification of important molecules that are deemed essential for HCV life cycle. The expanded set of putative virus and host-encoded targets has brought us one step closer to developing robust strategies for efficacious, pangenotypic, and well-tolerated medicines against HCV. Herein, we provide an overview of the development of various classes of virus and host-directed therapies that are currently in use along with others that are undergoing clinical evaluation.

The effects of differences in pspA alleles and capsular types on the resistance of Streptococcus pneumoniae to killing by apolactoferrin.

Pneumococcal surface protein A (PspA) is the only pneumococcal surface protein known to strongly bind lactoferrin on the bacterial surface. In the absence of PspA Streptococcus pneumoniae becomes more susceptible to killing by human apolactoferrin (apo-hLf), the iron-free form of lactoferrin. In the present study we examined diverse strains of S. pneumoniae that differed by 2 logs in their susceptibility to apo-hLf. Among these strains, the amount of apo-hLf that bound to cell surface PspA correlated directly with the resistance of the strain to killing by apo-hLf. Moreover examination of different pspA alleles on shared genetic backgrounds revealed that those PspAs that bound more lactoferrin conferred greater resistance to killing by apo-hLf. The effects of capsule on killing of pneumococci by apo-hLf were generally small, but on one genetic background, however, the lack of capsule was associated with 4-times as much apo-hLf binding and 30-times more resistance to killing by apo-hLf. Overall these finding strongly support the hypothesis that most of the variation in the ability of apo-hLf is dependent on the variation in the binding of apo-hLf to surface PspA and this binding is dependent on variation in PspA as well as variation in capsule which may enhance killing by reducing the binding of apo-hLf to PspA.

Meeting physical activity guidelines is associated with lower allostatic load and inflammation in Mexican Americans.

Examine the relationship between physical activity (PA) and allostatic load in Mexican-Americans as well as variations by gender. Self-reported PA as well as cardiovascular, metabolic and inflammatory markers were assessed in 330 Mexican-American adults in the Cameron County Hispanic Cohort (Brownsville, TX, USA). Dependent variables included total allostatic load, blood pressure, metabolic, and inflammatory scores. PA participation was categorized as sedentary, low, moderate, high, and by whether activity was sufficient to meet public health guidelines. Logistic regression analyses were conducted using cross-sectional data, and tested interaction effects of gender and PA. High active participants had lower allostatic load and inflammatory risk than sedentary participants. These relationships held for meeting versus not meeting guidelines. Males meeting guidelines were less likely to have high inflammation than other groups. The data did not suggest a dose-response association. These findings indicate that PA may reduce accumulation of allostatic load, highlighting the importance of a physically active lifestyle across the life span.

Infection and cellular defense dynamics in a novel 17ß-estradiol murine model of chronic human group B streptococcus genital tract colonization reveal a role for hemolysin in persistence and neutrophil accumulation.

Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17ß-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (10(6)-10(7) CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

Impaired CD4+ and T-helper 17 cell memory response to Streptococcus pneumoniae is associated with elevated glucose and percent glycated hemoglobin A1c in Mexican Americans with type 2 diabetes mellitus.

Individuals with type 2 diabetes are significantly more susceptible to pneumococcal infections than healthy individuals of the same age. Increased susceptibility is the result of impairments in both innate and adaptive immune systems. Given the central role of T-helper 17 (Th17) and T-regulatory (Treg) cells in pneumococcal infection and their altered phenotype in diabetes, this study was designed to analyze the Th17 and Treg cell responses to a whole heat-killed capsular type 2 strain of Streptococcus pneumoniae. Patients with diabetes demonstrated a lower frequency of total CD+T-cells, which showed a significant inverse association with elevated fasting blood glucose. Measurement of specific subsets indicated that those with diabetes had, low intracellular levels of interleukin (IL)-17, and lower pathogen-specific memory CD4+ and IL-17+ cell numbers. No significant difference was observed in the frequency of CD4+ and Th17 cells between those with and without diabetes. However, stratification of data by obesity indicated a significant increase in frequency of CD4+ and Th17 cells in obese individuals with diabetes compared with nonobese individual with diabetes. The memory CD+T-cell response was associated inversely with both fasting blood glucose and percent glycated hemoglobin A1c. This study demonstrated that those with type 2 diabetes have a diminished pathogen-specific memory CD4+ and Th17 response, and low percentages of CD+T-cells in response to S. pneumoniae stimulation.

Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression.

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications.