Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.     

Rapid genotyping of common deficient thiopurine S-methyltransferase alleles using the DNA-microchip technique

Thiopurine drugs are metabolized, in part, by S-methylation catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low or undetectable TPMT activity are at high risk of severe, potentially fatal hematopoietic toxicity when they are treated with standard doses of thiopurines. As human TPMT activity is controlled by a common genetic polymorphism, it is an excellent candidate for the clinical application of pharmacogenetics. Here, we report a new molecular approach developed to detect point mutations in the TPMT gene that cause the loss of TPMT activity. A fluorescently labeled amplified DNA is hybridized with oligonucleotide DNA probes immobilized in gel pads on a biochip. The specially designed TPMT biochip can recognize six point mutations in the TPMT gene and seven corresponding alleles associated with TPMT deficiency: TPMT*2; TPMT*3A, TPMT*3B, TPMT*3C, TPMT*3D, TPMT*7, and TPMT*8. The effectiveness of the protocol was tested by genotyping 58 samples of known genotype. The results showed 100% concordance between the biochip-based approach and the established PCR protocol. The genotyping procedure is fast, reliable and can be used for rapid screening of inactivating mutations in the TPMT gene. The study also provides the first data on the frequency of common TPMT variant alleles in the Russian population, based on a biochip analysis of 700 samples. TPMT gene mutations were identified in 44 subjects; genotype *1/*3A was most frequent.     

Temporomandibular joint loading generated during bilateral static bites at molars and premolars

The aim of this study was to investigate the features of the loading vectors of the temporomandibular joint (TMJ) generated during bilateral static bites at the molars and at the premolars, and to determine the major factors affecting the difference between the two loading vectors. We computed the subjects' estimated and theoretical minimum TMJ loadings under the two different bite conditions by applying the subjects' bite-force and electromyographic (EMG) data to a two-dimensional (2D) standard model of the jaw based on a rigid-body spring model of the TMJ. For a molar bite, (1) the estimated loading vector was not equal to its theoretical minimum; (2) the TMJ-loading/bite-force ratio, describing the proportion of TMJ loading, was relatively small, 0.477 on average; and (3) the estimated loading vector pointed in the direction of the central part of the articular disk's intermediate zone. For a premolar bite, on the other hand, (1) the estimated loading vector was nearly equal to its theoretical minimum; (2) the TMJ-loading/bite-force ratio was relatively large, 0.904 on average; and (3) the estimated loading vector pointed at the superior portion of the articular disk's intermediate zone. The differences between the TMJ-loading vectors for molar and premolar bites originated primarily from changes in the bite-point location.     

The association between cumulative periodontal disease and stroke history in older adults

BACKGROUND: Since the late 1980s, several studies have been conducted to investigate the relationship between periodontal disease and ischemic stroke. The purpose of this study is to investigate the relationship of periodontal disease to the self-reported history of stroke in the elderly (60 years of age and older) by examining the data of the Third National Health and Nutrition Examination Survey (NHANES III). METHODS: Data from NHANES III, a large population-based cross-sectional survey of the United States, were used for this study. Because 1,563 of the 5,123 subjects in the study were edentulous, and periodontal disease is a major cause of tooth loss, it was necessary to account for edentulousness in the statistical analysis to avoid bias. Hence, a new index called the periodontal health status (PHS) index was developed to address this problem. Two measures of PHS were developed: PHS I, based on the median percentage of sites with >/=2 mm clinical attachment loss (CAL), and PHS II, based on the median percentage of sites with >/=3 mm CAL. Multiple logistic regression analysis was used to test for the association of PHS with stroke history. Two types of a multiple logistic regression model were fit: 1) logistic regression modeling with adjustment for age and tobacco use only; and 2) logistic regression modeling with adjustment of all statistically significant confounders. RESULTS: Based on multiple logistic regression analysis of PHS with adjustment for age and tobacco use only, completely edentulous elderly adults (PHS Class 5) and partially edentulous (teeth in one arch) elderly adults with appreciable clinical attachment loss (PHS Class 4) were significantly more likely to have a history of stroke compared to dentate adults (teeth in both arches) without appreciable clinical attachment loss (PHS Class 1). When multiple logistic regression models were fit with adjustment of all significant confounders, no statistically significant association was found between PHS and stroke. CONCLUSIONS: Based on the results of this study, there is evidence of an association between cumulative periodontal disease, based on PHS, and a history of stroke. However, it is unclear whether cumulative periodontal disease is an independent risk factor for stroke or a risk marker for the disease.     

Enamel matrix derivative and coronal flaps to cover marginal tissue recessions

BACKGROUND: Correcting recession defects is one of the goals of periodontal therapy, and the efficacy and predictability of the various techniques are important considerations for both patients and clinicians. Several reports have examined the outcome of gingival recession treatment by means of coronally positioned flaps (CPF) and enamel matrix derivative (EMD). The purpose of this study was to clinically evaluate the use of EMD in association with CPF to cover localized gingival recessions compared to CPF alone. METHODS: Twenty-two patients with Miller Class I or II gingival recessions >2 mm were included. One recession from each patient was treated in the study. Two treatments were randomly assigned: coronally positioned flap with EMD (test) and coronally positioned flap alone (control). Clinical parameters measured at baseline and 1, 6, and 12 months included gingival index, plaque index, probing depth, clinical attachment level, vertical and horizontal recession, and width of keratinized gingiva. RESULTS: At 12 months, both treatment modalities showed significant root coverage, gain in clinical attachment, and gain in width of keratinized gingiva (P <0.05). Vertical recessions were reduced from 2.68 +/- 1.63 mm to 0.36 +/- 0.60 mm in the test group and from 2.31 +/- 1.52 mm to 0.90 +/- 0.95 mm in the control group. Horizontal recessions decreased from 4.27 +/- 2.06 mm to 0.77 +/- 0.87 mm in the test group and from 3.68 +/- 1.91 mm to 1.72 +/- 1.31 mm in the control group. Changes in keratinized gingiva went from 3.81 +/- 1.95 mm to 4.63 +/- 2.15 mm in the test group and from 3.31 +/- 1.81 mm to 3.27 +/- 1.80 mm in the control group. When both treatments were compared at 12 months, there was a significant difference in vertical tooth coverage and gain in keratinized gingiva in favor of the experimental group (P <0.05). The average percentage of root coverage for test and control groups was 88.6% and 62.2%, respectively. CONCLUSIONS: The coronally positioned flap alone or with EMD is an effective procedure to cover localized gingival recessions. The addition of EMD significantly improves the amount of root coverage.     

Use of perftoran emulsion to decrease allogeneic blood transfusion in cardiac surgery: clinical trial

Perflurocarbon emulsions (PFC) have the capacity of transporting oxygen through the bloodstream and may be safe and effective alternatives to allogeneic blood transfusions during surgical procedures. Perftoran was the PFC used in a randomized clinical trial conducted at Hospital de Especialidades Centro Medico La Raza, Mexico City. The clinical trial took a sample group, n = 30, of patients that were scheduled for elective cardiac valvuloplasty surgery in combination with preoperative acute normovolemic hemodilution and an inspiratory oxygen fraction (FI02) of 1.0. The participants were randomly divided into a Control group (n = 15) and a Perftoran (PFC) group (n = 15). The PFC group had significantly higher intraoperative PaO2 levels and needed less allogeneic red blood cell packs than the Control group. There were no complications or deaths in either group. These results suggest that Perftoran is safe, efficacious, and reduces the need for allogeneic blood and blood derivatives in patients undergoing cardiac surgery.     

Effect of mechanical and antiseptic therapy on peri-implant mucositis: an experimental study in monkeys

OBJECTIVES: This experiment was performed to evaluate clinically and histologically the effect of mechanical therapy with or without antiseptic therapy on peri-implant mucositis lesions in nine cynomolgus monkeys. MATERIAL AND METHODS: Two ITI titanium implants were inserted into each side of the mandibles. After 90 days of plaque control and soft tissue healing, a baseline clinical examination was completed. Peri-implant lesions were induced by placing silk ligatures and allowing plaque to accumulate for 6 weeks. The clinical examination was then repeated, and the monkeys were randomly assigned to three treatment groups: group A, mechanical cleansing only; group B, mechanical cleansing and local irrigation with 0.12% chlorhexidine (CHX) and application of 0.2% CHX gel; and group C, control, no treatment. The implants in treatment groups A and B were treated and maintained according to the assigned treatment for two additional months. At the end of the maintenance period, a final clinical examination was performed and the animals were sacrificed for biopsies. RESULTS: The mean probing depths (PD) values at mucositis were: 3.5, 3.7, and 3.4 mm, and clinical attachment level (CAL) = 3.8, 4.1, and 3.9 mm for treatment groups A, B and C, respectively. The corresponding values after treatment were: PD = 1.7, 2.1, and 2.5 mm, and CAL=2.6, 2.6, and 3.1 mm. ANOVA of mean changes (Delta) in PD and CAL after treatment showed no statistical difference between the treatment groups. Comparison of the mean changes in PD and CAL after treatment yielded statistical differences between the control and treatment groups P < 0.01. According to the t-test, no statistical difference was found between treatment groups A and B for the PD reduction but there was a significant difference for the CAL change, P < 0.03. Group A had significantly more recession and less CAL gain than group B. Non-parametric tests yielded no significant differences in modified plaque index (mPlI) and gingival index (GI) after treatment between both treatment groups. Frequencies and percent distributions of the mPlI and GI scores changed considerably for both treatment groups when compared with the changes in the control group after treatment. With regard to the histological evaluation, no statistical differences existed between the treatments for any linear measurement. The proportion of inflammation found in the mucosal tissues of the control implants was greater than the one found for both treatment groups, P < 0.01. More importantly, both treatment groups showed a similar low proportion of inflammation after 2 months of treatment. CONCLUSIONS: Within the limitations of this experiment, and considering the supportive plaque control rendered, it can be concluded that for pockets of 3-4 mm: (1) mechanical therapy alone or combined with CHX results in the clinical resolution of peri-implant mucositis lesions, (2) histologically, both treatments result in minimal inflammation compatible with health, and (3) the mechanical effect alone is sufficient to achieve clinical and histologic resolution of mucositis lesions.     

Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF

MurF is a key enzyme in the biosynthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. This enzyme has not been extensively exploited as a drug target, possibly due to the difficulty in obtaining one of the substrates, UDP-MurNAc-L-Ala-gamma-D-Glu-meso-diaminopimelate, which is usually purified from bacteria. We have identified putative inhibitors of Escherichia coli MurF by a binding assay, thus bypassing the need for substrate. Inhibition of enzymatic activity was demonstrated in a high-performance liquid chromatography-based secondary assay with UDP-MurNAc-L-Ala-gamma-D-Glu-diaminopimelate substrate prepared in a novel way by using muropeptide ligase enzyme to add UDP-MurNAc to synthetic L-Ala-gamma-D-Glu-diaminopimelate; the substrate specificity of muropeptide ligase for peptides containing L-Lys in place of diaminopimelate was also investigated. Using the muropeptide ligase-generated MurF substrate, a thiazolylaminopyrimidine series of MurF enzyme inhibitors with 50% inhibitory concentration values as low as 2.5 microM was identified.