BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dubé syndrome: a new series of 50 families and a review of published reports.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHDS) (MIM 135150) is an autosomal dominant predisposition to the development of follicular hamartomas (fibrofolliculomas), lung cysts, spontaneous pneumothorax, and kidney neoplasms. Germline mutations in BHD are associated with the susceptibility for BHDS. We previously described 51 BHDS families with BHD germline mutations. OBJECTIVE: To characterise the BHD mutation spectrum, novel mutations and new clinical features of one previously reported and 50 new families with BHDS. METHODS: Direct bidirectional DNA sequencing was used to screen for mutations in the BHD gene, and insertion and deletion mutations were confirmed by subcloning. We analysed evolutionary conservation of folliculin by comparing human against the orthologous sequences. RESULTS: The BHD mutation detection rate was 88% (51/58). Of the 23 different germline mutations identified, 13 were novel consisting of: four splice site, three deletions, two insertions, two nonsense, one deletion/insertion, and one missense mutation. We report the first germline missense mutation in BHD c.1978A>G (K508R) in a patient who presented with bilateral multifocal renal oncocytomas. This mutation occurs in a highly conserved amino acid in folliculin. 10% (5/51) of the families had individuals without histologically confirmed fibrofolliculomas. Of 44 families ascertained on the basis of skin lesions, 18 (41%) had kidney tumours. Patients with a germline BHD mutation and family history of kidney cancer had a statistically significantly increased probability of developing renal tumours compared to patients without a positive family history (p = 0.0032). Similarly, patients with a BHD germline mutation and family history of spontaneous pneumothorax had a significantly increased greater probability of having spontaneous pneumothorax than BHDS patients without a family history of spontaneous pneumothorax (p = 0.011). A comprehensive review of published reports of cases with BHD germline mutation is discussed. CONCLUSION: BHDS is characterised by a spectrum of mutations, and clinical heterogeneity both among and within families.

Mass vaccination: solutions in the skin.

The skin is populated with Langerhans cells, thought to be efficient, potent antigen-presenting cells, that are capable of inducing protective immunity by targeting antigen delivery to the skin. Delivery to the skin may be accomplished by active delivery such as intradermal injection, use of patches or a combination of a universal adjuvant patch with injections. The robust immunity induced by skin targeting can lead to dose sparing, novel vaccines and immune enhancement in populations with poorly responsive immune systems, such as the elderly. Vaccine delivery with patches (transcutaneous immunization), may allow self-administration, ambient temperature stabilization and ease of storage for stockpiling, leading to a new level of efficient vaccine distribution in times of crisis such as a bioterror event or pandemic influenza outbreak. The use of an adjuvant (immunostimulant) patch with injected vaccines has been shown in clinical studies to enhance the immune response to an injected vaccine. This can be used for dose sparing in pandemic influenza vaccines in critically short supply or immune enhancement for poor responders to flu vaccines such as the elderly. Transcutaneous immunization offers a unique safety profile, as adjuvants are sequestered in the skin and only delivered systemically by Langerhans cells. This results in an excellent safety profile and allows use of extremely potent adjuvants. The combination of the skin immune system, safe use of potent adjuvants and ease of delivery suggests that skin delivery of vaccines can address multiple unmet needs for mass vaccination scenarios.

Fumarate hydratase enzyme activity in lymphoblastoid cells and fibroblasts of individuals in families with hereditary leiomyomatosis and renal cell cancer.

BACKGROUND: Hereditary leiomyomatosis and renal cell cancer (HLRCC) is the autosomal dominant heritable syndrome with predisposition to development of renal cell carcinoma and smooth muscle tumours of the skin and uterus. OBJECTIVE: To measure the fumarate hydratase (FH) enzyme activity in lymphoblastoid cell lines and fibroblast cell lines of individuals with HLRCC and other familial renal cancer syndromes. METHODS: FH enzyme activity was determined in the whole cell, cytosolic, and mitochondrial fractions in 50 lymphoblastoid and 16 fibroblast cell lines including cell lines from individuals with HLRCC with 16 different mutations. RESULTS: Lymphoblastoid cell lines (n = 20) and fibroblast cell lines (n = 11) from individuals with HLRCC had lower FH enzyme activity than cells from normal controls (p<0.05). The enzyme activity in lymphoblastoid cell lines from three individuals with mutations in R190 was not significantly different from individuals with other missense mutations. The cytosolic and mitochondrial FH activity of cell lines from individuals with HLRCC was reduced compared with those from control cell lines (p<0.05). There was no significant difference in enzyme activity between control cell lines (n = 4) and cell lines from affected individuals with other hereditary renal cancer syndromes (n = 22). CONCLUSIONS: FH enzyme activity testing provides a useful diagnostic method for confirmation of clinical diagnosis and screening of at-risk family members.

Novel mutations in FH and expansion of the spectrum of phenotypes expressed in families with hereditary leiomyomatosis and renal cell cancer.

BACKGROUND: Hereditary leiomyomatosis and renal cell cancer (HLRCC; OMIM 605839) is the predisposition to develop smooth muscle tumours of the skin and uterus and/or renal cancer and is associated with mutations in the fumarate hydratase gene (FH). Here we characterise the clinical and genetic features of 21 new families and present the first report of two African-American families with HLRCC. METHODS: Using direct sequencing analysis we identified FH germline mutations in 100% (21/21) of new families with HLRCC. RESULTS: We identified 14 germline FH mutations (10 missense, one insertion, two nonsense, and one splice site) located along the entire length of the coding region. Nine of these were novel, with six missense (L89S, R117G, R190C, A342D, S376P, Q396P), one nonsense (S102X), one insertion (111insA), and one splice site (138+1G>C) mutation. Four unrelated families had the R58X mutation and five unrelated families the R190H mutation. Of families with HLRCC, 62% (13/21) had renal cancer and 76% (16/21) cutaneous leiomyomas. Of women FH mutation carriers from 16 families, 100% (22/22) had uterine fibroids. Our study shows that expression of cutaneous manifestations in HLRCC ranges from absent to mild to severe cutaneous leiomyomas. FH mutations were associated with a spectrum of renal tumours. No genotype-phenotype correlations were identified. CONCLUSIONS: In combination with our previous report, we identify 31 different germline FH mutations in 56 families with HLRCC (20 missense, eight frameshifts, two nonsense, and one splice site). Our FH mutation detection rate is 93% (52/56) in families suspected of HLRCC.

A simple method for studying whole sections of rice grain.

We developed a new and simple method to collect sections of a whole brown rice kernel for investigation of histological properties. A single kernel of rice was dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. During sectioning of the blocks using a rotary microtome, we used a special adhesive tape to collect and place the sections on slides so they remained flat. The use of the adhesive tape technique combined with autofluorescence characteristics allowed us to visualize cell walls throughout an entire longitudinal or transverse section of a whole rice kernel. We obtained scanning electron microscopy images of the sections to determine section quality.

Effect of transcutaneous immunization with co-administered antigen and cholera toxin on systemic and mucosal antibody responses in sheep.

Direct application of antigens to skin together with an adjuvant, a procedure called transcutaneous immunization (TCI), can induce systemic immune responses in mice, humans, cats and dogs. In previous studies we found that cholera toxin (CT) applied topically on unbroken skin induces systemic antibody and lymphocyte proliferative responses in sheep. The current study examined whether concurrent administration of CT and tetanus toxoid (TT) delivered transcutaneously could induce specific antibody responses to both antigens in sheep. Antibodies to both TT and CT were induced by TCI although antibody titres in serum to TT were higher in sheep receiving TT plus alum by intramuscular injection (n=5) than TT plus CT by TCI (n=5). The ratio of IgG1/IgG2 antibody to TT in serum was near unity, and the route of immunization, TCI versus injection, did not influence this ratio. In contrast, the ratio of IgG1/IgG2 antibody differed significantly between the two antigens, TT and CT, delivered by TCI, with a higher proportion of IgG1 antibody in serum to CT than TT. Antibody to TT was detected in lung washes from TCI and injection groups, with IgG1 predominating over IgG2 in both groups. IgA antibodies to CT and TT were detected in sera of CT and TT-immunized groups respectively but in lung washes IgA antibody to TT was detected only in the injection group. Results show that TCI induced systemic antibody responses to CT and the co-administered antigen TT, whereas no evidence was obtained for mucosal IgA responses following TCI.

Comorbid genetic diseases, von Hippel-Lindau disease and spinocerebellar ataxia type 2, confounding the diagnosis of cerebellar dysfunction in an adolescent.

The authors report a 15-year-old female who presented with difficulties in ambulation as well as difficulties with balance and penmanship. She had a known genetic risk of von Hippel-Lindau (VHL; MIM 193300) disease, with a unique VHL mutation, but had no tumors of the brain or spine to explain her symptoms. Laboratory analysis of peripheral blood lymphocytes was targeted at genetic loci associated with ataxic disorders. Allelic expansion of the ataxin-2 gene was identified. Spinocerebellar ataxia type 2 (SCA2) was diagnosed as a comorbid genetic condition in this patient.

Generation of adenoviruses encoding the herpes simplex virus vhs gene: a novel strategy to generate adenoviruses expressing genes toxic to producer cells.

Adenovirus vectors expressing suicide genes represent a promising approach for cancer gene therapy. We wanted to determine whether the virion host shutoff (vhs) gene of herpes simplex virus could be used as a suicide gene for gene therapy of glioblastomas. The vhs gene was cloned downstream of the glial fibrillary acidic protein promoter to direct tissue-specific expression, and recombinant adenoviruses were generated in 293 cells. Viruses, which contained the vhs gene but did not express it, could readily be isolated; however, we were unable to plaque purify viruses that expressed vhs protein. We constructed a derivative of 293 cells expressing an antisense RNA to the vhs gene and used them to generate adenovirus vectors that express vhs protein. These cells were used to complement vhs-expressing adenoviruses through three rounds of plaque purification. This approach could be generalized to produce adenovirus vectors expressing other toxic transgenes.

Transcutaneous immunization: T cell responses and boosting of existing immunity.

Transcutaneous immunization (TCI) is a novel immunization strategy by which antigen and adjuvant are applied topically to intact, hydrated skin to induce potent antibody and cell-mediated immune responses specific for both the antigen and the adjuvant. Using tetanus toxoid as a model antigen, we examined the T cell response to tetanus toxoid after topical immunization with a variety of adjuvants. TCI readily induced systemic antigen specific T cell responses with a mixed Th1/Th2 phenotype but with a Th2 bias. We also investigated whether priming by the intramuscular route, which is known to induce T cell memory, could be followed by a boosting immunization on the skin to induce secondary responses. TCI could augment existing immunity, but interestingly, this strategy induced potent responses only if the antibody titer was low at the time of TCI boosting. These and previous observations suggest that TCI follows known immunological principles that govern other routes of vaccine delivery. Furthermore, booster immunization using tetanus toxoid may provide a useful model for further development of important patch and formulation concepts for TCI, and act as an early candidate for validating product feasibility of TCI in humans.

Transcutaneous immunization: an emerging route of immunization and potent immunostimulation strategy.

Transcutaneous immunization (TCI) has emerged recently as a new method of vaccination that uses the skin. The simplicity of a patch-based immunization may obscure the potency of this strategy for immunostimulation because TCI allows the safe use of a wide variety of potent adjuvants. It is thought that these adjuvants activate Langerhans cells in the skin, which migrate to the draining lymph to orchestrate robust systemic immune responses. TCI represents a novel combination using established knowledge relating to skin penetration, the potency of adjuvant-based immunostimulation, and data showing that Langerhans cells are highly desirable targets because of their antigen-presenting cell function. The near-term challenge will be to take this promising insight into successful product development.

Transcutaneous immunization: a human vaccine delivery strategy using a patch.

Transcutaneous immunization, a topical vaccine application, combines the advantages of needle-free delivery while targeting the immunologically rich milieu of the skin. In animal studies, this simple technique induces robust systemic and mucosal antibodies against vaccine antigens. Here, we demonstrate safe application of a patch containing heat-labile enterotoxin (LT, derived from Escherichia coli) to humans, resulting in robust LT-antibody responses. These findings indicate that TCI is feasible for human immunization, and suggest that TCI may enhance efficacy as well as improve vaccine delivery.

Transcutaneous immunization of domestic animals: opportunities and challenges.

Transcutaneous immunization (TCI), the topical application of antigen and adjuvant directly onto intact skin, can safely and effectively elicit systemic immune responses in mice and humans against a variety of antigens. This novel method of vaccine delivery has the potential to provide a safe and convenient method by which vaccines may be delivered to elicit protective immunity in domestic animals. To date, however, immune responses induced by TCI in companion and production animals has not been reported. In this report, we demonstrate that TCI may be widely applicable to many animals. Immune responses elicited by TCI require further optimization for each antigen and species, and success may depend upon the structure and composition of the skin of the target species. The prospect of TCI as a practical and broadly applicable approach to vaccination in veterinary medicine is discussed in the context of these challenges.

Transcutaneous immunization with bacterial ADP-ribosylating exotoxins, subunits, and unrelated adjuvants.

We have recently described a needle-free method of vaccination, transcutaneous immunization, consisting of the topical application of vaccine antigens to intact skin. While most proteins themselves are poor immunogens on the skin, we have shown that the addition of cholera toxin (CT), a mucosal adjuvant, results in cellular and humoral immune responses to the adjuvant and coadministered antigens. The present study explores the breadth of adjuvants that have activity on the skin, using diphtheria toxoid (DTx) and tetanus toxoid as model antigens. Heat-labile enterotoxin (LT) displayed adjuvant properties similar to those of CT when used on the skin and induced protective immune responses against tetanus toxin challenge when applied topically at doses as low as 1 microg. Interestingly, enterotoxin derivatives LTR192G, LTK63, and LTR72 and the recombinant CT B subunit also exhibited adjuvant properties on the skin. Consistent with the latter finding, non-ADP-ribosylating exotoxins, including an oligonucleotide DNA sequence, as well as several cytokines (interleukin-1beta [IL-1beta] fragment, IL-2, IL-12, and tumor necrosis factor alpha) and lipopolysaccharide also elicited detectable anti-DTx immunoglobulin G titers in the immunized mice. These results indicate that enhancement of the immune response to topical immunization is not restricted to CT or the ADP-ribosylating exotoxins as adjuvants. This study also reinforces earlier findings that addition of an adjuvant is important for the induction of robust immune responses to vaccine antigens delivered by topical application.

Mosaicism in von Hippel-Lindau disease: lessons from kindreds with germline mutations identified in offspring with mosaic parents.

von Hippel-Lindau disease (VHL [MIM 193300]) is a heritable autosomal dominant multiple-neoplastic disorder with high penetrance. It is characterized by brain and spinal-cord hemangioblastomas, retinal angiomas, clear-cell renal carcinoma, neuroendocrine tumors and cysts of the pancreas, pheochromocytomas, endolymphatic-sac tumors, and papillary cystadenomas of the epididymis and broad ligament. Although most index cases have a positive family history of VHL, some do not and may represent de novo cases. Cases without a family history of VHL may or may not have a germline mutation in their VHL tumor-suppressor gene. We present two cases of VHL mosaicism. In each of two families, standard testing methods (Southern blot analysis and direct sequencing) identified the germline mutation in the VHL gene of the offspring, but not in their clinically affected parent. Additional methods of analysis of the affected parents' blood detected the VHL-gene mutation in a portion of their peripheral blood lymphocytes. In one case, detection of the deleted allele was by FISH, and, in the second case, the 3-bp deletion was detected by conformational sensitive gel electrophoresis and DNA sequencing of cloned genomic DNA. Mosaicism in VHL is important to search for and recognize when an individual without a family history of VHL has VHL. Patients diagnosed without family histories of the disease have been reported in as many as 23% of kindreds with VHL. Identification of individuals potentially mosaic for VHL will affect counseling of families, and these individuals should themselves be included in clinical screening programs for occult disease.

Induction of systemic immune responses in sheep by topical application of cholera toxin to skin.

Cholera (and related) toxins (CT) when applied topically on unbroken skin induce systemic immune responses in mice, a procedure called transcutaneous immunization (TCI). The current study examined the capacity for TCI to induce systemic immune responses in sheep. Three groups (n=5 per group) were immunized at day 0 (priming) and day 28 (boosting) with 250 microg of CT in water by TCI, with 25 microg of CT in alum by intramuscular injection, or not immunized. Serum samples were taken at days 0, 28, 42, 56 and 70 after immunization for measurement of CT-specific IgG as well as CT-specific IgG1, IgG2, IgA and IgM antibodies by ELISA. After immunization, IgG, IgG1 and IgG2 antibody in immunized groups were significantly higher than in the control group, and boosting further increased these titres. IgG, IgG1 and IgG2 in the injection group were significantly higher than in the TCI group. There was a preponderance of IgG1 antibody, relative to IgG2, in both immunized groups. CT-specific IgA and IgM were detected in both immunized groups. Lymphocyte proliferation to CT was measured at day 90. A CT-specific lymphocyte proliferative response (stimulation index>2) was detected in all sheep from the injection group, in two sheep from the TCI group and in none of the controls. Results demonstrated that TCI induces primary and secondary antibody responses and specific proliferative responses to CT in sheep.

Sorption of volatile flavor compounds by microcellular cereal starch.

Wheat starch based microcellular foam (MCF) forms of (dry) starch possess a significant percentage of micropores in the range of 5-14 A. The present study confirmed earlier preliminary studies that MCF starch (in a 0.25-1.0 mm diameter bead form) is effective in sorbing and lowering the headspace partial pressure of many volatile compounds in a manner similar (although less efficient) to that exhibited by other microcavity sorbants such as charcoal. It was found that the proportion of polar compounds sorbed was much greater than the proportion of nonpolar compounds. A major portion of the sorbed volatile compound was readily displaced from the MCF microcellular starch by the addition of water. These properties make this form of edible starch a potential useful carrier of flavor compounds for dried foods.

Principles of transcutaneous immunization using cholera toxin as an adjuvant.

Transcutaneous immunization is a novel strategy for immunization employing topical application of antigen and adjuvant to the skin surface and resulting in detectable antigen/adjuvant specific IgG in plasma and mucosal secretions. In this study we show that transcutaneous immunization with cholera toxin (CT) as an adjuvant can be used in several inbred mouse strains with varying H-2 major histocompatibility complex genes (C57BL/6 (H-2(b)), BALB/c (H-2(d)), and C3H (H-2(k))). Although the primary anti-CT antibody responses reflected previously described MHC restriction patterns for this protein, the differences were overcome after two booster immunizations. Potent antibody responses against hen egg lysozyme and/or diphtheria toxoid were observed using CT as adjuvant. We also demonstrate that the unshaved dorsal or ventral surface of the ear can be effectively used for transcutaneous immunization and that gentle swabbing with alcohol increases the magnitude of the host immune response. Together these data further our understanding of the principles governing this new platform technology and support its integration into novel and existing human vaccine strategies.

Clinical and genetic characterization of pheochromocytoma in von Hippel-Lindau families: comparison with sporadic pheochromocytoma gives insight into natural history of pheochromocytoma.

PURPOSE: Families with von Hippel-Lindau disease have variable risk of pheochromocytoma. Patients with von Hippel-Lindau disease and pheochromocytoma identified by screening can have no characteristic signs or symptoms. Families with von Hippel-Lindau disease were screened and followed to describe the natural history of von Hippel-Lindau pheochromocytoma, and to correlate these findings with von Hippel-Lindau germline mutation. MATERIALS AND METHODS: Between 1988 and 1997, 246 individuals with von Hippel-Lindau disease were identified (von Hippel-Lindau group). Between August 1990 and June 1997, 26 consecutive patients with sporadic pheochromocytoma were evaluated (sporadic group). RESULTS: A total of 64 patients with von Hippel-Lindau disease had manifestations of pheochromocytoma, including 33 newly diagnosed during screening at the National Institutes of Health and 31 previously treated (93 adrenal and 13 extra-adrenal pheochromocytomas). Germline von Hippel-Lindau gene missense mutation was associated with extra-adrenal pheochromocytoma, younger age at presentation and the only patient with metastases. Of the 33 newly diagnosed patients with von Hippel-Lindau disease 4 had pheochromocytoma 2 times (37 pheochromocytomas) during followup. Of these pheochromocytomas 35% (13 of 37) were associated with no symptoms, normal blood pressure and normal catecholamine testing. Comparison of urinary catecholamines in the von Hippel-Lindau and sporadic groups demonstrated increased epinephrine, metanephrines and vanillylmandelic acid in the sporadic group. Analysis of urinary catecholamine excretion in the von Hippel-Lindau and sporadic groups together demonstrated a correlation between tumor size, and urinary metanephrines, vanillylmandelic acid, norepinephrine, epinephrine and dopamine. In 12 patients without signs or symptoms of pheochromocytoma 17 newly diagnosed pheochromocytomas were followed for a median of 34.5 months without morbidity. Median tumor doubling time was 17 months. CONCLUSIONS: Von Hippel-Lindau gene missense mutation correlated with the risk of pheochromocytoma in patients with von Hippel-Lindau disease. These findings support a von Hippel-Lindau disease clinical classification, wherein some families are at high risk for manifestations of pheochromocytoma. Von Hippel-Lindau disease pheochromocytomas identified by screening were smaller and less functional than sporadic pheochromocytomas.

Transcutaneous immunization with bacterial ADP-ribosylating exotoxins as antigens and adjuvants.

Transcutaneous immunization (TCI) is a new technique that uses the application of vaccine antigens in a solution on the skin to induce potent antibody responses without systemic or local toxicity. We have previously shown that cholera toxin (CT), a potent adjuvant for oral and nasal immunization, can induce both serum and mucosal immunoglobulin G (IgG) and IgA and protect against toxin-mediated mucosal disease when administered by the transcutaneous route. Additionally, CT acts as an adjuvant for coadministered antigens such as tetanus and diphtheria toxoids when applied to the skin. CT, a member of the bacterial ADP-ribosylating exotoxin (bARE) family, is most potent as an adjuvant when the A-B subunits are present and functional. We now show that TCI induces secondary antibody responses to coadministered antigens as well as to CT in response to boosting immunizations. IgG antibodies to coadministered antigens were also found in the stools and lung washes of immunized mice, suggesting that TCI may target mucosal pathogens. Mice immunized by the transcutaneous route with tetanus fragment C and CT developed anti-tetanus toxoid antibodies and were protected against systemic tetanus toxin challenge. We also show that bAREs, similarly organized as A-B subunits, as well as the B subunit of CT alone, induced antibody responses to themselves when given via TCI. Thus, TCI appears to induce potent, protective immune responses to both systemic and mucosal challenge and offers significant potential practical advantages for vaccine delivery.