Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy.

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

Toward developing a universal treatment for fungal disease using radioimmunotherapy targeting common fungal antigens.

BACKGROUND: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall. METHODS: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls. RESULTS: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing. CONCLUSION: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Molar-incisor-hypomineralisation: a literature review.

BACKGROUND: Molar-Incisor-Hypomineralisation (MIH) is a qualitative defect of 1-4 first permanent molars with or without the maxillary and mandibular permanent incisors. It seems to have been recognised first in the 1970s and prevalence varies between 2.8% and 25%, dependent upon the study. METHODS: The dental literature was searched using a number of key terms entered into MEDLINE, the reference list of each paper as located was examined for further papers that had been missed in the initial search. RESULTS: The review of the literature showed that teeth that are affected indicate a systemic cause at around the time of birth; investigators have put forward a number of possible causes; asthma, pneumonia, upper respiratory tract infections, otitis media, antibiotics, dioxins in mother's milk, tonsillitis and tonsillectomy and exanthamatous fevers of childhood. However, at the present time the aetiology remains unclear. Treatment of the affected permanent first molars can include restorations using adhesive intra-coronal restorations to extra-coronal restorations (e.g. preformed metal crowns). There is little evidence to support one option over another. In severe cases extraction at the optimum time may be the best option; allowing the permanent second molars to come forwards. There is little improvement in affected anterior teeth with microabrasion and direct or indirect composite resin restorations may be appropriate in some children. Ultrastructural and biochemical make up of MIH affected teeth seem to have been investigated less than other areas. CONCLUSION: It is important that children with MIH are diagnosed as early as possible and managed appropriately; this will involve multidisciplinary input.

Case report: scurvy in an epileptic child on a ketogenic diet with oral complications.

BACKGROUND: Epilepsy is a symptom of cerebral dysfunction, where there is a sudden and disorganised discharge of electrical activity from a group of neurones, producing symptoms that range from sensory absences to convulsive movements and unconsciousness. Fasting is recognised as reducing the frequency of epileptic seizures in difficult to control patients. The ketogenic diet is a high fat, low carbohydrate and adequate protein diet that mimics the biochemical effects of fasting. It is deficient in some essential elements that require supplementation. CASE REPORT: A 9-year old girl with learning difficulties, developmental delay and refractory epilepsy was placed on the ketogenic diet in 2003. Prior to starting the diet she had had as many as 12 tonic seizures/day, with prolonged periods of non-convulsive status epilepticus. Subsequent to being placed on the diet, the frequency of her seizures reduced markedly; there were long periods during which she had none. In late 2006, the patient inhaled a primary molar. This was retrieved by emergency bronchoscopy and at the same time the remaining primary teeth were extracted. Three weeks later she was admitted to hospital with low-grade fever, persistently bleeding sockets, oedema of her hands and feet, a petechial rash and bruising. A differential diagnosis included: liver disease, bleeding dyscrasia, oncological pathology or scurvy. The most striking finding amongst a number of investigations was a vitamin C level of 0.7 micromol/l (Deficiency: < 11 micromol/l). Accordingly a diagnosis of scurvy was made. TREATMENT: The patient was prescribed ascorbic acid 500 mg twice/day. Three weeks later the patient's vitamin C level was 141.5 micromol/l; the dose was therefore reduced to 250 mg once/day. FOLLOW-UP: At two-month review, the signs and symptoms of scurvy had resolved. CONCLUSION: Inhaling a tooth and scurvy are both rare occurrences. Paediatric dentists should be aware of the possible implications of a ketogenic diet.

Treatment of aesthetic problems in paediatric dentistry.

Aesthetic problems in childhood and adolescence can have a significant effect on psychosocial development and interaction with peers. Abnormalities of shape, size, colour and structure of the whole or part of the anterior dentition of children can lead to such problems. This article outlines the most appropriate options for the clinical management of different aetiological categories of aesthetic problems, and develops a hierarchy of intervention that should be pursued in a logical order until a satisfactory cosmetic outcome is achieved. These techniques are readily available to most dental practitioners and there is no reason for a child to have to wait until late adolescence for treatment. Dent Update 2003; 30: 307-313 CLINICAL RELEVANCE: Aesthetic problems in children and adolescents may be treated by a variety of techniques.

Antibody-guided alpha radiation effectively damages fungal biofilms.

The use of indwelling medical devices--pacemakers, prosthetic joints, catheters--is rapidly growing and is often complicated by infections with biofilm-forming microbes that are resistant to antimicrobial agents and host defense mechanisms. We investigated for the first time the use of microbe-specific monoclonal antibodies (MAbs) as delivery vehicles for targeting biofilms with cytocidal radiation. MAb 18B7 (immunoglobulin G1 [IgG1]), which binds to capsular polysaccharides of the human pathogenic fungus Cryptococcus neoformans, penetrated cryptococcal biofilms, as shown by confocal microscopy. When the alpha radiation-emitter 213-Bismuth ((213)Bi) was attached to MAb 18B7 and the radiolabeled MAb was added to C. neoformans biofilms, there was a 50% reduction in biofilm metabolic activity. In contrast, when the IgM MAb 13F1 labeled with (213)Bi was used there was no penetration of the fungal biofilm and no damage. Unlabeled 18B7, (213)Bi-labeled nonspecific MAbs, and gamma and beta types of radiation did not have an effect on biofilms. The lack of efficacy of gamma and beta radiation probably reflects the radioprotective properties of polysaccharide biofilm matrix. Our results indicate that C. neoformans biofilms are susceptible to treatment with antibody-targeted alpha radiation, suggesting a novel option for the prevention or treatment of microbial biofilms on indwelling medical devices.

Retrospective analysis of factors influencing the eruption of delayed permanent incisors after supernumerary tooth removal.

AIM: This was to assess the predictability of eruption of delayed permanent incisors after supernumerary removal and creation of adequate space, in relation to: root maturity, degree of vertical impaction, and degree of angulation of impaction. METHODS: The dental records of children with supernumerary teeth delaying the eruption of permanent incisors were analysed. The type of a supernumerary tooth, its location and position were recorded, along with the stage of root maturation, angulation and vertical distance of impaction of the permanent incisor. At the initial surgery, the unerupted supernumerary tooth and any retained primary incisors were removed. The unerupted permanent incisor was not exposed. If necessary, the maxillary primary canines were removed to create sufficient space for eruption of the delayed permanent tooth. A secondary surgical procedure was planned after 18 months if there was no significant progress of the permanent tooth towards eruption. STATISTICS: All data were entered onto a Microsoft Excel spread sheet and analysed using Fisher's Exact Tests throughout due to the small numbers. RESULTS: Sixty-six supernumerary teeth were removed, 22 from boys and 44 from girls with ages ranging from 6 to 10 years 6 months at the time of surgery. Primary canines were extracted in 59.1% of cases. Spontaneous eruption occurred in 89.4% of delayed permanent teeth. The mean time to eruption was 9.2 months (median = 7 months). There was no statistically significant association between tooth eruption and root maturity or the degree of vertical impaction. There was an association between eruption and the degree of the angle of impaction of the permanent incisor (p<0.05). CONCLUSION: The majority of delayed permanent teeth erupt spontaneously if sufficient space is available or created at the time of removal of the unerupted supernumerary. The angulation of impaction of the permanent incisor is associated with a delay in eruption.

Radiological studies reveal radial differences in the architecture of the polysaccharide capsule of Cryptococcus neoformans.

The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans is an important virulence factor, but relatively little is known about its architecture. We applied a combination of radiological, chemical, and serological methods to investigate the structure of this polysaccharide capsule. Exposure of C. neoformans cells to gamma radiation, dimethyl sulfoxide, or radiolabeled monoclonal antibody removed a significant part of the capsule. Short intervals of gamma irradiation removed the outer portion of the cryptococcal capsule without killing cells, which could subsequently repair their capsules. Survival analysis of irradiated wild-type, acapsular mutant, and complemented mutant strains demonstrated that the capsule contributed to radioprotection and had a linear attenuation coefficient higher than that of lead. The capsule portions remaining after dimethyl sulfoxide or gamma radiation treatment were comparable in size, 65 to 66 microm3, and retained immunoreactivity for a monoclonal antibody to glucuronoxylomannan. Simultaneous or sequential treatment of the cells with dimethyl sulfoxide and radiation removed the remaining capsule so that it was not visible by light microscopy. The capsule could be protected against radiation by either of the free radical scavengers ascorbic acid and sorbitol. Sugar composition analysis of polysaccharide removed from the outer and inner parts of the capsule revealed significant differences in glucuronic acid and xylose molar ratios, implying differences in the chemical structure of the constituent polysaccharides. Our results provide compelling evidence for the existence of two zones in the C. neoformans capsule that differ in susceptibility to dimethyl sulfoxide and radiation and, possibly, in packing and composition.

Feasibility of radioimmunotherapy of experimental pneumococcal infection.

Streptococcus pneumoniae is an important cause of community-acquired pneumonia, meningitis, and bacteremia. The problem of pneumococcal disease is exacerbated by increasing drug resistance. Furthermore, patients with impaired immunity are at high risk for invasive pneumococcal infections. Thus, there is an urgent need for new approaches to antimicrobial therapy. Antibody therapies take advantage of the specificity and high affinity of the antigen-antibody interaction to deliver antibacterial compounds to a site of infection in the form of naked or conjugated antibodies. We have recently established that radioimmunotherapy (RIT) can be used to treat experimental fungal infections in mice. In the present study, we investigated the feasibility of applying a RIT approach to the treatment of S. pneumoniae infection by evaluating the susceptibility of S. pneumoniae to radiolabeled antibody in vitro and in an animal infection model. For the specific antibody carrier, we used human monoclonal antibody D11, which binds to pneumococcal capsular polysaccharide 8. We have selected the alpha particle emitter (213)Bi as the radionuclide for conjugation to the antibody. Incubation of serotype 8 S. pneumoniae with (213)Bi-D11 resulted in dose-dependent killing of bacteria. RIT of S. pneumoniae infection in C57BL/6 mice showed that 60% more mice survived in the (213)Bi-D11-treated group (80 micro Ci) than in the untreated group (P < 0.01). The treatment did not cause hematological toxicity, as demonstrated by platelet counts. This feasibility study establishes that RIT can be applied to the treatment of bacterial infections.

Evaluation of acute hematologic and long-term pulmonary toxicities of radioimmunotherapy of Cryptococcus neoformans infection in murine models.

We evaluated acute hematological and long-term pulmonary toxicity of radioimmunotherapy in murine models of Cryptococcus neoformans infection. Activities up to 250 microCi were well tolerated by healthy A/JCr mice for (213)Bi-18B7 and (188)Re-18B7 monoclonal antibodies. In infected mice, doses up to 150 microCi produced only transient toxicity. The lungs of treated mice had no evidence of radiation fibrosis.

Factors affecting outcomes of traumatically extruded permanent teeth in children.

PURPOSE: The objectives of the present study were to determine the prevalence of residual extrusion, pulpal necrosis, and resorption for extruded permanent teeth and to establish the effect of presentation and treatment factors. METHODS: Seventy-two traumatically extruded permanent incisors were studied at the Departments of Paediatric Dentistry in Belfast, Newcastle upon Tyne, and Glasgow. The mean age of the patients was 10.1 years (range=6 to 18 years). Clinical and radiographic outcomes were analyzed and related to presenting and treatment factors. RESULTS: The initial degree of extrusion was moderate for 46 teeth (64%), and the median delay prior to repositioning was 3 hours (range=1 to 168 hours). Pulp necrosis occurred in 31 teeth (43%), residual extrusion was present in 16 teeth (23%), and inflammatory resorption occurred in 11 teeth (15%). Residual extrusion was significantly associated with a delay in repositioning the tooth, pulpal necrosis was significantly more common in teeth with closed apices and in severely extruded teeth, and inflammatory resorption was more common after pulpal necrosis. CONCLUSIONS: Residual extrusion could be minimized by earlier presentation and repositioning. The risk of pulpal necrosis is greatest for severely extruded teeth and for those with closed apices.

The success of inhalation sedation for comprehensive dental care within the Community Dental Service.

OBJECTIVES: To assess the outcomes of treatment with nitrous oxide/oxygen inhalation sedation (IS). To relate these to the age and previous dental experience of the child and the experience of the operator. To provide base-line information and identify training needs. METHODS: A retrospective examination of the clinical records of all children treated with IS within the Community Dental Service of Harrow and Hillingdon NHS Trust (HHHT) over a 3-month period was made. Personal details and previous dental experience were recorded. The outcome of the planned treatment was identified. RESULTS: Two hundred and eleven sets of records were reviewed from eight clinicians. The average age of the children was 7.2 years. Treatment plans were successfully completed in 83.9% of cases. Records showed that 18.5% of the children had previously had general anaesthesia (GA) for dental treatment, 27.5% had received IS and 5.2% had no previous dental experience. Of the 'failed' treatments, 50% were under 7 years of age and 31.3% were referred for GA. There was no difference in the proportion of failures in relation to the experience of the operator. CONCLUSION: This review shows that inhalation sedation with nitrous oxide/oxygen is a very successful adjunct to the clinical management of children within the Community Dental Service.

Infraocclusion: a case report with an unexpected outcome.

The management of infraoccluded primary molars can pose some difficult clinical questions: when to treat? What treatment to provide? Why treat? How long to wait and watch? The definition and aetiology of infraoccluded primary molars is discussed. Some of the sequelae of different treatment options are described. A case is presented of the management of a ten and a half year old girl with a severely infraoccluded maxillary left second primary molar. The initial treatment plan included surgical removal of both the infraoccluded primary molar and the permanent successor, which was impacted high in the maxilla. However, the sudden unexpected eruption of the permanent tooth allowed a successful non-surgical outcome.

Dental disease and caries related microflora in children with dystrophic epidermolysis bullosa.

PURPOSE: The purpose of this study was to investigate dental caries, bacterial dental plaque, gingivitis and caries related oral microflora in children with predominantly autosomal recessive Dystrophic Epidermolysis Bullosa (DEB). METHODS: Thirty children with DEB from The Great Ormond Street Hospital for Children and 31 control children matched for age, gender and ethnicity were included in the study. RESULTS: The main findings were: 1. A significantly greater mean dmft in the DEB children (p < 0.05). 2. A significantly greater mean plaque score for the DEB children for both the primary (p < 0.001) and permanent teeth (p < 0.02) compared with the control children. 3. A significantly greater mean gingivitis score for the DEB children for both the primary (p < 0.002) and permanent teeth (p < 0.0001) compared with the control children. 4. A significantly greater salivary total anaerobic count for the control children compared with the DEB children (p < 0.001). CONCLUSIONS: The results reflect the difficulties that children with DEB have with basic oral hygiene procedures combined with slow oral clearance.

Control of synthesis and positioning of a Caulobacter crescentus flagellar protein.

The Caulobacter crescentus flagellum is assembled during a defined time period in the cell cycle. Two genes encoding the major components of the flagellar filament, the 25K and the 27.5K flagellins, are expressed coincident with flagellar assembly. A third gene, flgJ, is also temporally regulated. The synthesis of the product of flgJ, the 29K flagellin, occurs prior to the synthesis of the other flagellin proteins. We demonstrate here that the time of initiation of flgJ expression is independent of chromosomal location but is dependent upon cis-acting sequences present upstream of the flgJ structural gene. Evidence that there is transcriptional control of flgJ expression includes the following: (1) The initial appearance of flgJ message was coincident with the onset of 29K flagellin protein synthesis, and (2) expression of an NPT II reporter gene driven by the flgJ promoter was temporally correct. Post-transcriptional regulation might contribute to the control of expression, because the flgJ mRNA persisted for a longer period of time than did the synthesis of the 29K protein. The 29K flagellin was found only in the progeny swarmer cell after cell division. In a mutant strain that failed to assemble a flagellum, the 29K flagellin still segregated to the presumptive swarmer cell, demonstrating that positioning of the protein is independent of filament assembly. Analysis of a chimeric flgJ-NPT II transcriptional fusion showed that the flgJ regulatory sequences do not control the segregation of the 29K flagellin to the swarmer cell progeny, suggesting that correct segregation depends on the protein product.

Effect of 2, 3-diphosphoglycerate on the solubility of deoxy-sickle hemoglobin.

We have examined the effect of 2, 3-diphosphoglycerate (DPG) on the solubility of deoxy-sickle hemoglobin (deoxy-Hb S) under conditions such that concentration, pH, and osmolarity of deoxy-Hb S solutions approached physiological. The range of DPG/Hb molar ratios encompassed the extremes found for this ratio in erythrocytes from individuals with sickle cell anemia. After monomer-polyer equilibrium had been established, the phases were separated by centrifugation and assayed for concentrations of Hb and DPG. DPG had no effect on the solubility of deoxy-Hb S. Furthermore, at DPG/Hb molar ratios less than one, there was no preferential incorporation of deoxy-Hb S containing bound DPG into polymers. At DPG/Hb molar ratios greater than one, concentrations of free DPG in monomer and polymer phases were virtually identical. Thus, under the specified equilibrium conditions, DPG is not a determining factor in the polymerization of deoxy-Hb S.