Shaper Mirza

Crimean Congo-Haemorrhagic Fever treated with oral ribavirin

Crimean-Congo Haemorrhagic Fever (CCHF) is an often-lethal haemorrhagic fever caused by a tick-borne virus. There are no published data on ribavirin treatment of CCHF-infected patients, despite established in-vitro and in-vivo sensitivity. We report three health workers--two surgeons and a hospital worker--infected with CCHF virus in Pakistan who were treated with oral ribavirin 4 g/day for four days, then 2.4 g/day for six days. Intravenous ribavirin was unavailable. All three patients were severely ill with low platelet and white-cell counts, raised aspartate transaminase and evidence of impaired haemostasis. Based on published reports, all had an estimated probability of death of 90% or more. The patients became afebrile, and their haematological and biochemical abnormalities returned to normal within 48 h of ribavirin treatment; all made a complete recovery, and developed IgG and IgM antibody to CCHF virus. Our experience with ribavirin treatment is encouraging, but does not constitute evidence of efficacy. Given the difficulties in gathering adequate treatment data, we propose a consensus protocol for both intravenous and oral treatment of CCHF. This protocol could be distributed to key medical personnel in areas endemic for CCHF and used to provide a firm basis for effective treatment recommendations.

Unsafe injections and the transmission of hepatitis B and C in a periurban community in Pakistan

Following reports of frequent deaths associated with jaundice and chronic liver disease among adults in a periurban community of Karachi, Pakistan, an investigation was conducted to evaluate the relationship between injections and viral hepatitis infections, to identify the reasons why patients received frequent injections, and to observe the injection practices employed in clinics. Two hundred and three adult patients were interviewed as they left each of the 18 area clinics. Practitioners were interviewed and three consecutive injections were observed at each clinic. Eighty-one per cent of patients received an injection on the day of the interview. Of the 135 patients who provided a serum sample, 59 (44%) had antibodies against hepatitis C virus and 26 (19%) had antibodies against hepatitis B virus. Patients who received more injections were more likely to be infected with hepatitis C. If oral and injected medications were equally effective, 44% of patients preferred injected medication. None of the practitioners knew that hepatitis C could be transmitted by injections. Non-sterile syringes and needles that had been used earlier in the day on other patients were used for 94% of the observed injections. Interventions to limit injections to those which are safe and clinically indicated are needed to prevent injection-associated infections in Pakistan and other low-income countries.

Type 2-diabetes is associated with elevated levels of TNF-alpha, IL-6 and adiponectin and low levels of leptin in a population of Mexican Americans: A cross-sectional study

The goal of the study was to determine the association between diabetes and inflammation in clinically diagnosed diabetes patients. We hypothesized that low-grade inflammation in diabetes is associated with the level of glucose control. Using a cross-sectional design we compared pro- and anti-inflammatory cytokines in a community-recruited cohort of 367 Mexican Americans with type 2-diabetes having a wide range of blood glucose levels. Cytokines (IL-6, TNF-α, IL-1β, IL-8) and adipokines (adiponectin, resistin and leptin) were measured using multiplex ELISA. Our data indicated that diabetes as whole was strongly associated with elevated levels of IL-6, leptin, CRP and TNF-α, whereas worsening of glucose control was positively and linearly associated with high levels of IL-6, and leptin. The associations remained statistically significant even after controlling for BMI and age (p=0.01). The association between TNF-α, however, was attenuated when comparisons were performed based on glucose control. Strong interaction effects between age and diabetes and BMI and diabetes were observed for IL-8, resistin and CRP. The cytokine/adipokine profiles of Mexican Americans with diabetes suggest an association between low-grade inflammation and quality of glucose control. Unique to in our population is that the chronic inflammation is accompanied by lower levels of leptin.

Characterization of binding of human lactoferrin to pneumococcal surface protein A

ABSTRACT Human lactoferrin is an iron-binding glycoprotein that is particularly prominent in exocrine secretions and leukocytes and is also found in serum, especially during inflammation. It is able to sequester iron from microbes and has immunomodulatory functions, including inhibition of both complement activation and cytokine production. This study used mutants lacking pneumococcal surface protein A (PspA) and PspC to demonstrate that the binding of human lactoferrin to the surface of Streptococcus pneumoniae was entirely dependent on PspA. Lactoferrin bound both family 1 and family 2 PspAs. Binding of lactoferrin to PspA was shown by surface colocalization with PspA and was verified by the lack of binding to PspA-negative mutants. Lactoferrin was expressed on the body of the cells but was largely absent from the poles. PspC showed exactly the same distribution on the pneumococcal surface as PspA but did not bind lactoferrin. PspAs binding site for lactoferrin was mapped using recombinant fragments of PspA of families 1 and 2. Binding of human lactoferrin was detected primarily in the C-terminal half of the α-helical domain of PspA (amino acids 167 to 288 of PspA/Rx1), with no binding to the N-terminal 115 amino acids in either strain. The interaction was highly specific. As observed previously, bovine lactoferrin bound poorly to PspA. Human transferrin did not bind PspA at all. The binding of lactoferrin to S. pneumoniae might provide a way for the bacteria to interfere with host immune functions or to aid in the acquisition of iron at the site of infection.

Expansion of epidemic dengue viral infections to Pakistan.

OBJECTIVES Antibodies to dengue viruses have occasionally been reported in individuals in Pakistan, but the frequency of occurrence of dengue infection in Pakistan is unclear. The first confirmed dengue hemorrhagic fever outbreak in Pakistan occurred in 1994. In October 1995, the authors investigated an outbreak of a febrile illness among employees of a construction contractor at a power generation plant in Baluchistan, Pakistan, to determine the cause of illness and recommend appropriate preventive measures. METHODS The work site and living arrangements were inspected, a questionnaire was administered, and serum samples were collected from all consenting contractor employees and their families if they lived at the camp. Sera were analyzed for IgM against dengue virus, using an enzyme-linked immunosorbent assay. RESULTS Interviews were conducted with 76 persons (mean age, 42y); 95% were men. Forty-two persons (55%) reported having experienced fever, headache, or myalgia in the preceding 6 weeks. Fifty-seven subjects (75%) had IgM antibodies against at least one dengue serotype; 45 subjects (59%) had IgM antibodies against dengue serotype 2. CONCLUSION This was an outbreak of dengue fever due to multiple serotypes of dengue virus. This confirms that epidemic dengue infection was present in southern Pakistan for 2 consecutive years.

Pneumococcal Surface Protein A Inhibits Complement Deposition on the Pneumococcal Surface by Competing with the Binding of C-Reactive Protein to Cell-Surface Phosphocholine

In the presence of normal serum, complement component C3 is deposited on pneumococci primarily via the classical pathway. Pneumococcal surface protein A (PspA), a major virulence factor of pneumococci, effectively inhibits C3 deposition. PspA’s C terminus has a choline-binding domain that anchors PspA to the phosphocholine (PC) moieties on the pneumococcal surface. C-reactive protein (CRP), another important host defense molecule, also binds to PC, and CRP binding to pneumococci enhances complement C3 deposition through the classical pathway. Using flow cytometry of PspA+ and PspA− strains, we observed that the absence of PspA led to exposure of PC, enhanced the surface binding of CRP, and increased the deposition of C3. Moreover, when the PspA− mutant was incubated with a pneumococcal eluate containing native PspA, there was decreased deposition of CRP and C3 on the pneumococcal surface compared with incubation with an eluate from a PspA− strain. This inhibition was not observed when a recombinant PspA fragment, which lacks the choline-binding region of PspA, was added to the PspA− mutant. Also, there was much greater C3 deposition onto the PspA− pneumococcus when exposed to normal mouse serum from wild-type mice as compared with that from CRP knockout mice. Furthermore, when CRP knockout mouse serum was replenished with CRP, there was a dose-dependent increase in C3 deposition. The combined data reveal a novel mechanism of complement inhibition by a bacterial protein: inhibition of CRP surface binding and, thus, diminution of CRP-mediated complement deposition.

Serine Protease PrtA from Streptococcus pneumoniae Plays a Role in the Killing of S. pneumoniae by Apolactoferrin

ABSTRACT It is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, including Streptococcus pneumoniae. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease, prtA, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (P < 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin kills Streptococcus pneumoniae requires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci.

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.

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.

Modified Opsonization, Phagocytosis, and Killing Assays To Measure Potentially Protective Antibodies against Pneumococcal Surface Protein A

ABSTRACT The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant α-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA.