The ␣ 6 integrin subunit participates in the formation of both ␣ 6 ␤ 1 and ␣ 6 ␤ 4 laminin receptors, which have been reported to play an important role in cell adhesion and migration and in morphogenesis. In squamous epithelia, the ␣ 6 ␤ 4 heterodimer is the crucial component for the assembly and stability of hemidesmosomes. These anchoring structures are ultrastructurally abnormal in patients affected with junctional epidermolysis bullosa with pyloric atresia (PA-JEB), a recessively inherited blistering disease of skin and mucosae characterized by an altered immunoreactivity with antibodies specific to integrin ␣ 6 ␤ 4. In this report, we describe the first mutation in the ␣ 6 integrin gene in a PA-JEB patient presenting with generalized skin blistering, aplasia cutis, and defective expression of integrin ␣ 6 ␤ 4. The mutation (791delC) is a homozygous deletion of a single base (C) leading to a frameshift and a premature termination codon that results in a complete absence of ␣ 6 polypeptide. We also describe the DNA-based prenatal exclusion of the disease in this family at risk for recurrence of PA-JEB. Our results demonstrate that, despite the widespread distribution of the ␣ 6 integrin subunit, lack of expression of the ␣ 6 integrin chain is compatible with fetal development, and results in a phenotype indistinguishable from that caused by mutations in the ␤ 4 chain, which is expressed in a more limited number of tissues. ( J. Clin. Invest. 1997. 99:2826-2831.)

Herlitz junctional epidermolysis bullosa (H-JEB) providesdeposited into the extracellular matrix. Re-expression of a promising model for somatic gene therapy of heritable laminin-5 induced cell spreading, nucleation of hemidesmechano-bullous disorders. This genodermatosis is mosomal-like structures and enhanced adhesion to the culcaused by the lack of laminin-5 that results in absence of ture substrate. Organotypic cultures performed with the hemidesmosomes (HD) and defective adhesion of squamtransduced keratinocytes, reconstituted epidermis closely ous epithelia. To establish whether re-expression of lamiadhering to the mesenchyme and presenting mature heminin-5 can restore assembly of the dermal-epidermal attachdesmosomes, bridging the cytoplasmic intermediate filament structures lacking in the H-JEB skin, we corrected the ments of the basal cells to the anchoring filaments of the genetic mutation hindering expression of the ␤3 chain of basement membrane. Our results provide the first evilaminin-5 in human H-JEB keratinocytes by transfer of a dence of phenotypic reversion of JEB keratinocytes by laminin ␤3 transgene. The transduced keratinocytes synsomatic gene therapy and demonstrate that genetic treatthesized a recombinant ␤3 polypeptide that assembled ment of the mild forms of skin blistering diseases and other with the endogenous laminin ␣3 and ␥2 chains into a bioinherited extracellular matrix pathologies is a realistic goal. logically active laminin-5 that was secreted, processed and

Laminin-5 is the major adhesion ligand of epithelial cells. Mutations in the three genes (LAMA3, LAMB3, LAMC2) encoding the laminin-5 chains cause junctional epidermolysis bullosa, a clinically and genetically heterogeneous blistering skin disease. Here, we describe a non-Herlitz junctional epidermolysis bullosa patient, compound heterozygote for two novel mutations affecting the LAMC2 gene. The mutation in the paternal allele is a de novo splice site mutation (522-1G-->A) that results in in-frame skipping of exon 4 and synthesis of a mutated gamma2 polypeptide (gamma2Delta4) carrying a 33 amino acid deletion within the N-terminal domain V. The maternal mutation is a one base pair insertion (3511insA) in the 3' terminal exon of LAMC2 resulting in a frameshift and a premature termination codon. Mutation 3511insA is predicted to lead to the synthesis of a gamma2 polypeptide (gamma2t) disrupted in its alpha-helical C-terminal structure and truncated of the last 25 amino acids. Keratinocytes isolated from the patient's skin showed a markedly decreased level of gamma2 chain mRNA and secreted scant amounts of laminin-5, which undergoes physiologic proteolytic processing. To investigate the biologic function of the laminin-5 molecules synthesized by the patient, mutant gamma2 cDNAs were transiently expressed in gamma2-null keratinocytes. Transfection of the gamma2Delta4 cDNA resulted in restoration of laminin-5 deposition onto the culture substrate, which demonstrates that the gamma2 polypeptides carrying a deletion in domain V, upstream of the gamma2 proteolytic cleavage site, are assembled into native laminin-5 that is secreted and extracellularly processed. In contrast, transfection of a mutant cDNA expressing the gamma2t chain failed to restore laminin-5 immunoreactivity, which indicates that integrity of the gamma2 C-terminal amino acid sequences is required for laminin-5 assembly. These results correlate for the first time a functional alteration in a laminin-5 domain with a mild junctional epidermolysis bullosa phenotype.

Sialyl Lewis x (sLex) oligosaccharides have been shown to be present in counterreceptors for L-selectin. We and others have previously shown that high endothelial cells in lymph nodes and at sites of inflammation express sLex. Here we show that also cultured human umbilical vein endothelial cells (HUVEC) express sLex on their cell surface. This oligosaccharide is formed by sequential action of alpha 2,3-sialyl- (alpha 2,3-ST) and alpha 1,3-fucosyltransferases (alpha 1,3-FT) on N-acetyllactosamine. At least two of the several alpha 2,3-ST and four of the several alpha 1,3-FT are present in HUVEC. In functional assays both alpha 2,3-ST and alpha 1,3-FT activities were observed in HUVEC lysates with exogenous lactosamine and sialyllactosamine acceptors, leading to the generation of the sialyllactosamine and sLex sequences, respectively. TNF stimulation increased the level of mRNA expression of FT VI, and the alpha 1,3-FT activity in HUVEC. Taken together these data show that endothelial cells express sLex and that they possess mRNA as well as enzyme activities of several alpha 2,3-ST and alpha 1,3-FT necessary in the final steps of sLex synthesis. Furthermore, inflammatory cytokines such as TNF can enhance transferase activities relevant in generating putative L-selectin counterreceptors.

With reference to the clinical need for simple, potent and safe antiretroviral regimens, Lopinavir/ ritonavir + tenofovir (LPV/r+TDF) two-drug initial regimen was studied for efficacy and safety in HIV-infected patients. Methods: Kalead was a prospective, randomized, open-label, 72-week trial comparing LPV/r+TDF versus LPV/r + two (non-TDF) NRTIs in HIV-infected adults with HIV-RNA >400 copies/mL and any CD4 count. Primary endpoint was the proportion of subjects with HIV-RNA <50 copies/mL at week 72. Results: 152 subjects were randomized. Eleven (15.3%) subjects in the dual therapy arm and seven (8.8%) in the triple therapy arm who did not achieve HIV-RNA <50 copies/mL at least twice prior to and including week 24 were discontinued per protocol (p=0.21). Overall discontinuations were 41.7% and 43.8% in the dual therapy and triple therapyarms. At week 72, 51.4% and 52.5% of subjects in the dual therapy and triple therapy arms had HIV-RNA <50 copies/mL (p=0.89, ITT, NC=F). In an on-treatment analysis, 87.2% and 93.0% of subjects in the dual therapy and triple therapy arms had a HIV-RNA <50 copies/mL (p=0.47). Over 72 weeks of therapy, mean CD4 count increases were greater in the dual therapy arm (+332 cells/mm 3 vs +234 cells/mm 3 , p=0.01). Adherence, overall incidence of adverse events, drugrelated adverse events, and Grade I-IV laboratory abnormalities were comparable between the two arms. Conclusions: A two-drug regimen of LPV/r+TDF suggests sufficient safety and efficacy warranting further investigation. However, high discontinuation rate and study design limitations restrict overall interpretation.