<Emphasis Type="Italic">In Vivo</Emphasis> Toxicity and Immunological Characterization of Detoxified Recombinant Botulinum Neurotoxin Type A

Purpose

A double-mutant E224A/E262A full-length botulinum neurotoxin (BoNT) Type A with structural similarity to native BoNT/A but lacking the endopeptidase activity provides an ideal surrogate for testing pharmacokinetics and immunochemical characteristics of BoNT.

Methods

We determined lethality (LD₅₀) of deactivated recombinant botulinum neurotoxin (drBoNT/A) to be 24.0 μg by intraperitoneal route (i.p). The polypeptide drBoNT/A labeled with near infra-red dye 800 (NIR 800) was used to examine its distribution to different organs using whole body imaging when administered to mice via intravenous (i.v) or i.p route. Also, drBoNT/A was used to evaluate its immunogenicity in Balb/C mice model.

Results

drBoNT/A was found to be highly immunogenic when tested under various in vivo conditions in Balb/C mice model. For the first time we have demonstrated that a full length 150 kDa drBoNT/A, by administering via inhalation route in mice model, has evoked both circulating immunoglobulin levels of IgG and secretory IgA at the mucosal surface. The immunoglobulin levels were sufficient enough to protect against the challenge dose of native BoNT toxin in mice model. Tissue distribution of drBoNT/A seems to be similar to that of native toxin.

Conclusions

Based on the characteristics described in this report this nontoxic holotoxin protein will assist us to explore the window of opportunity available for therapeutic treatment in case of unnatural poisoning, and also it can be an effective vaccine candidate.

     

Troubleshooting electromagnetic interference in a patient with centrifugal flow left ventricular assist device and subcutaneous implantable cardioverter defibrillator

A 25‐year‐old man with severe nonischemic dilated cardiomyopathy underwent subcutaneous implantable cardioverter defibrillator (S‐ICD) implant and subsequently underwent HeartWare ventricular assist device (HVAD) placement. Postoperative interrogation revealed both primary and secondary S‐ICD vectors inappropriately regarded sinus rhythm as “noise,” and the alternate vector significantly undersensed sinus rhythm. The S‐ICD was reinterrogated using high‐resolution capture to visually confirm EMI with a dominant frequency in both the primary and secondary vectors of 46.67 Hz that fell within the S‐ICD operational range of 9–60 Hz. The 46.67 Hz frequency correlated with the HVAD operational speed of 2,800 RPM. The HVAD pump speed was increased from 2,800 to 3,000 RPM, resulting in a dominant frequency of 50 Hz. The notch filter is nonprogrammable in S‐ICDs. However, the built‐in filter is 50 Hz for countries in European time zones as opposed to 60 Hz in US time zones due to differences in the anticipated noise from electrical sources within each continent. Thus, the S‐ICD time zone was reprogrammed from EST to GMT, which reduced the notch filter from 60  to 50 Hz, resulting in S‐ICD successfully eliminating EMI when the patient was in a supine position. The EMI interference was still intermittently present in the upright patient position. This case demonstrates the utility of high‐resolution electrogram capture to identify the source and frequency of EMI in S‐ICD and offers a potential avenue to troubleshoot dominant frequency oversensing by changing the device time zone.     

Quantification of human skin barrier function and susceptibility to quantum dot skin penetration

Abstract In this study, we utilize in vivo human skin and a viable ex-vivo human skin model to investigate the effect of a commercial depilatory agent on barrier function. Tape stripping was used as a positive control. The magnitude of skin barrier was quantified by measuring transepidermal water loss values on in vivo human skin and transepithelial electrical resistance measurements and tissue histology on ex vivo skin. The susceptibility to carboxylated quantum dot penetration through ex vivo skin was investigated using fluorescent microcopy analysis of microtomed skin sections and flow cytometry to quantify quantum dot association with live epidermal cells. Results show that depilatory treatment modifies the outside-in barrier sufficiently to allow quantum dots to penetrate the stratum corneum but to a lesser extent than tape stripping. The implications of these finding are discussed.     

Early operable breast cancer in elderly women treated with an aromatase inhibitor letrozole as sole therapy

Background:

Primary endocrine therapy (PET) with aromatase inhibitors (AIs) is an option in elderly patients unfit for or unwilling to undergo surgery. We studied the outcome of patients treated with letrozole as PET.

Methods:

Patients with early oestrogen receptor (ER)/progesterone receptor (PR)-positive breast cancer treated with letrozole from February 2001 to September 2009 were reviewed. Inoperable and locally advanced tumours were excluded. Reasons for offering PET, response, survival, cause of death, time to initial and best response, fracture incidence, and late failure rates were studied.

Results:

In all, 104 patients received PET due to frailty (n=48), comorbidity (n=30), old age (n=9), and patient preference (n=17). Median follow-up was 56 months (4–106). Eighty-five cancers responded to letrozole (stable disease (SD, n=19), reduction in size (PR, n=42), and complete response ((CR), n=24)). Median survival was 51 months (4–103), time to initial response (PR/CR) 4.5 months (2–24), and time to best response 8.5 months (3–50). Letrozole was stopped in 25 patients due to progressive disease (n=19), side effects (n=5), and patient choice (n=1). Only 12 of 49 deaths were from breast cancer.

Conclusion:

Letrozole is a reasonable alternative in elderly women with early ER/PR-positive invasive breast cancer who are unfit or unwilling to undergo standard therapy.     

Construction and characterization of an auxotrophic ctxA mutant of O139 Vibrio cholerae

Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10⁶ and 10⁸ CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.     

Synthesis, characterization and pharmacological evaluation of amide prodrugs of ketorolac

Ketorolac (KC) suffers from the general side effects of NSAIDs, owing to presence of free carboxylic acid group. The study aimed to retard the adverse effects of gastrointestinal origin. Ten prodrugs of KC were synthesized by amidation with ethyl esters of amino acids, namely, glycine, l-phenylalanine, l-tryptophan, l-valine, l-isoleucine, l-alanine, l-leucine, l-glutamic acid, l-aspartic acid and beta-alanine. Purified synthesized prodrugs were characterized by m.p., TLC, solubility, partition coefficients, elemental analyses, UV, FTIR, NMR and MS. Synthesized prodrugs were subjected for biopharmaceutical studies, analgesic, anti-inflammatory activities and ulcerogenic index. Marked reduction of ulcerogenic index and comparable analgesic, anti-inflammatory activities were obtained in all cases as compared to KC. Among synthesized prodrugs, viz. AR-11, AR-19 and AR-20 showed excellent pharmacological response and encouraging hydrolysis rate both in SIF and in 80% human plasma. Prodrugs with increased aliphatic side chain length or introduction of aromatic substituent showed enhanced partition coefficient but diminished dissolution and hydrolysis rates. Such prodrugs can be considered for sustained release purpose.     

Importance of Adopting BMI Classifications Using Public Health Action Points to Delineate Obstetric Risk Factors Resulting in Worsening Obstetric Outcomes Among Asian Population

Aims

The aim of this study is to compare obstetric outcomes between overweight and class 1 obesity among pregnant women in their first pregnancy based on WHO’s BMI cut-offs and the potential public health action points identified by WHO expert consultations specific for high-risk population such as Asians.

Methods

This is a retrospective cohort review of data obtained from the Malaysian National Obstetrics and Gynaecology Registry between the year 2010 and year 2012. All women in their first pregnancy with a booking BMI in their first trimester were included in this study. The association between BMI classifications as defined by the WHO cut-offs and the potential public health action points identified by WHO expert consultations towards adverse obstetric outcomes was compared.

Results

A total of 88,837 pregnant women were included in this study. We noted that the risk of adverse obstetric outcomes was significantly higher using the public health action points identified by WHO expert consultations even among the overweight group as the risk of stillbirths was (OR 1.2; 95% CI 1.0,1.4), shoulder dystocia (OR 1.9; 95% CI 1.2,2.9), foetal macrosomia (OR 1.8; 95% CI 1.6,2.0), caesarean section (OR 1.9; 95% CI 1.8,2.0) and assisted conception (OR 1.9; 95% CI 1.6,2.1).

Conclusion

A specifically lower BMI references based on the potential public health action points for BMI classifications were a more sensitive predictor of adverse obstetric outcomes, and we recommend the use of these references in pregnancy especially among Asian population.