Three spontaneous H-2Db mutants are generated by genetic micro-recombination (gene conversion) events. Impact on the H-2-restricted immune responsiveness.

Sequence analysis of the mutant Dbm13, Dbm14, and Dbm24 genes indicate that they differ from the parental Db gene by 4, 1, and 8 nucleotides, respectively. The mutant sequences substituted into Dbm13 and Dbm24 are identical to those found in the Kb gene, at the homologous positions. Thus, similar to the Kb gene, the Db gene is able to undergo micro-recombination (gene conversion) events with other class I genes. Such data suggest that micro-recombination events could be an important mechanism for the diversification of all H-2 genes. The Db mutant products share a common theme: the alterations in all occur at amino acid residues whose side chains in the homologous class I HLA-A2 molecule project into the postulated peptide antigen-binding cleft, and hence, would be expected to alter the binding of foreign or self peptides. Due to such changes, the bm14 mouse has become a nonresponder in the CTL response to Moloney murine leukemia virus (M-MuLV), as the alteration of one amino acid residue at position 70 (a Gln to His) is sufficient to entirely abrogate the cell-mediated response to the virus. On the other hand, the bm13 mouse has shifted the major part of its M-MuLV restriction to Kb, a profound alteration in CTL responsiveness due to the alteration of three amino acids (Leu to Gln at 114, Phe to Tyr at 116, and Glu to Asp at 119) in a peptide stretch of beta-pleated sheet structure lining the bottom of the antigen-binding cleft. Thus, study of these mutants reveals that, in one step, micro-recombination at the genetic level has resulted at the protein level in profound changes in the immune response to viral infection. Such a mechanism operating at the population level can be a driving force during evolution for modulating the character of CTL immunity.

Comparison of the sensitivities of plantar nerve conduction techniques for early detection of diabetic sensory polyneuropathy.

The purpose of this study was to determine the most sensitive diagnostic test for nerve conduction study (NCS) of the foot for early detection of diabetic polyneuropathy. We compared the sensitivities for diagnosis of sensory polyneuropathy of four different nerve conduction techniques in the same nerves: nerve conduction studies of the medial plantar nerve with surface electrodes using three different techniques and a nerve conduction study of the digital and interdigital nerves of the foot using a near-nerve needle technique. In 25 patients with diabetic polyneuropathy with normal routine NCS, diagnosis of sensory neuropathy was confirmed by medial plantar NCS in 5 patients (20.0%) using Guiloff's method, in 5 patients (20.0%) using Ponsford's method and in 9 patients (36.0%) using Hemmi's method. In digital and interdigital NCS of the foot, a definite neuropathy pattern was observed in 15 patients (60.0%). The most common abnormality was low amplitude of sensory nerve action potential, indicating axonal degeneration. This study demonstrated that digital and interdigital NCS using the near-nerve needle technique is a more sensitive method for detection of early-stage diabetic polyneuropathy.

Immune response in mice that lack the interferon-gamma receptor.

Interferon-gamma (IFN-gamma) exerts pleiotropic effects, including antiviral activity, stimulation of macrophages and natural killer cells, and increased expression of major histocompatibility complex antigens. Mice without the IFN-gamma receptor had no overt anomalies, and their immune system appeared to develop normally. However, mutant mice had a defective natural resistance, they had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. Immunoglobulin isotype analysis revealed that IFN-gamma is necessary for a normal antigen-specific immunoglobulin G2a response. These mutant mice offer the possibility for the further elucidation of IFN-gamma-mediated functions by transgenic cell- or tissue-specific reconstitution of a functional receptor.

Functional role of type I and type II interferons in antiviral defense.

Mice lacking the known subunit of the type I interferon (IFN) receptor were completely unresponsive to type I IFNs, suggesting that this receptor chain is essential for type I IFN-mediated signal transduction. These mice showed no overt anomalies but were unable to cope with viral infections, despite otherwise normal immune responses. Comparison of mice lacking either type I or type II IFN receptors showed that, at least in response to some viruses, both IFN systems are essential for antiviral defense and are functionally nonredundant.

Muscular fatigue and decremental response to repetitive nerve stimulation in X-linked spinobulbar muscular atrophy.

BACKGROUND AND PURPOSE: We report decremental responses to repetitive nerve stimulation (RNS) in 11 patients diagnosed with X-linked spinobulbar muscular atrophy (X-SBMA). METHODS: The compound muscle action potential (CMAP) of the right abductor digiti minimi (ADM) and trapezius (TZ) in response to a 3-Hz stimulation of the ulnar nerve at the wrist and accessory nerve at the neck were recorded by surface electrodes. RESULTS: A decremental response to RNS was observed in 90.9% of the TZ muscle and 27.2% in the ADM muscle of patients with X-SBMA. CONCLUSION: These electrophysiological features of X-SBMA are considered to be useful for diagnosis of X-SBMA. Furthermore, the waning phenomena that mostly appeared in the TZ muscle and increment of CMAP in RNS after the exercise also suggest a unique manifestation in X-SBMA.

Increase in the frequency of p16INK4 gene inactivation by hypermethylation in lung cancer during the process of metastasis and its relation to the status of p53.

The p16INK4 gene, which is a tumor suppressor gene, is frequently altered in lung cancers. Hypermethylation of the promoter region of the p16INK4 gene seems to be the major mechanism through which p16INK4 become inactivated. Hypermethylation of the p16INK4 gene was reported to occur at an early stage in lung cancer. To determine whether the change in p16INK4 methylation status occurs at the late stage in the progression of primary lung cancers, we analyzed the primary and metastatic tumor tissues and normal lung samples from 29 cases of advanced lung cancer with distant metastasis. In each tissue sample, we analyzed the p16INK4 and p15INK4b genes for mutations and the methylation status of both genes using PCR-single strand conformation polymorphism, direct sequencing, and methylation-specific PCR analysis. We also analyzed a subset of the samples for p16INK4 protein expression. Genetic mutations in the coding region of the p16INK4 and p15INK4b genes were not found in any of the examined specimens. The promoter region of the p16INK4 gene was hypermethylated in the tumor samples of the primary or metastatic site of 37.0% (10 of 27) of the subjects. The promoter region of the p16INK4 gene was hypermethylated at both the primary and metastatic sites in two of the 10 cases and at only the metastatic site in 8 cases. By immunohistochemical analysis, we confirmed the presence of p16INK4 protein at the primary site of all cases in which the promoter region of the p16INK4 gene was hypermethylated at only the metastatic site. Interestingly, all 8 cases with a hypermethylated p16INK4 promoter region, at only the metastatic site, did not have p53 mutation. The results of this study indicate that tumor cells in which the p16INK4 gene has been inactivated by hypermethylation of the promoter region could have an advantage in progression and metastasis in non-small cell lung cancers, especially in the tumors with normal p53, and that the frequency of p16INK4 gene inactivation by hypermethylation could vary in clinical course.

The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures.

Adenovirus (AdV)-mediated gene expression of immune stimulators represents a valuable in vivo approach for gene therapy of human cancer. The expression level of the therapeutic gene is of crucial importance for the efficacy of this type of treatment. Entry of AdV is dependent on the primary adenovirus receptor CAR and the secondary AdV receptor identified earlier to be a member of the integrin family of surface molecules. We have analyzed 14 different human melanoma cell cultures from different stages together with one melanoma cell line for their AdV-mediated transduction and expression efficiency. Recombinant viruses at various concentrations were used for expression of the B7-1 costimulatory molecule under the control of different promoters and the expression levels of B7-1 were analyzed by flow cytometry. AdV-mediated IL-12 expression was measured using a commercial ELISA. Levels of transgene expression were compared with the expression levels of HCAR, the alpha(v)beta3 and alpha(v)beta5 integrins, and HLA class I. In 4 of 14 cell cultures tested, the presence of the primary virus receptor CAR was associated with the high transduction efficiency phenotype when using the B7-1- and IL-12-expressing viruses at a relatively low multiplicity of infection (MOI) of 50. Immunohistochemistry on cryosections from the original biopsies yielded a strong signal specific for CAR. In contrast, cell cultures expressing low or undetectable levels of CAR needed a 20- to 40-fold higher viral input to show comparable expression level of B7-1 or IL-12. Expression levels of the transgenes hardly varied when using different promoters and no association was observed with the presence or absence of HLA class I molecules or with the expression levels of integrins.

Comparative analysis of immunocritical melanoma markers in the mouse melanoma cell lines B16, K1735 and S91-M3.

The mouse melanoma cell lines B16, K1735 and Cloudman S91-M3 (and various sublines) are frequently used as melanoma models. Extensive comparative data of their immunological features are not available. In order to define the immunological profiles of these cell lines, relevant tumour markers were studied. S91-M3 melanoma cells constitutively expressed high levels of major histocompatibility complex (MHC) I, in contrast to K1735-M2 and B16-F1 cells. MHC II expression was restricted to B16-F1 cells following interferon-gamma treatment. Tyrosinase, tyrosinase-related protein-2 and gp100 were detected in B16-F1 and S91-M3 cells, but not in K1735-M2 cells. Constitutive surface expression and secretion of intercellular adhesion molecule-1 was found on S91-M3 cells. No substantial secretion of interleukin-10 could be detected. In contrast, low levels of latent transforming growth factor-beta were found in the cell supernatants of B16-F1 and K1735-M2 cells. The expression pattern of Fas, FasL and FLICE inhibitory protein was comparable in all three cell lines. Thus our findings indicate that each cell line presents a characteristic immunological profile, confirming that B16-F1 is an appropriate murine tumour model for tumours with low levels of MHC I but expressing melanoma-associated antigens. S91-M3 represents a complementary, more immunogenic model. In contrast, K1735-M2 does not seem to be an appropriate model for melanoma.

Upregulation of interleukin-12 receptor on peripheral blood mononuclear cells and HLA class I, HLA class II or ICAM-1 on melanoma cells by B7.1 and interleukin-12: a mechanism for immunostimulatory impact of melanoma cells adenovirally transfected with B7.1 and IL12?

Melanoma is an immunogenic tumour and may express both HLA class I and class II molecules. These can be recognized by cytotoxic T-cells. Melanoma cells can evade immunosurveillance due to the lack of co-stimulatory molecules such as B7.1 or B7.2. Interleukin-12 (IL12) exerts antitumour effects, and B7.1 and IL12 synergistically induce effective antitumour immunity. We investigated the immunostimulatory potential of melanoma cells adenovirally transduced with B7.1, IL12 or B7.1 plus IL12. We observed that: (i) melanoma cells transduced with B7.1 plus IL12 can elicit a strong proliferative response from peripheral blood mononuclear cells (PBMCs); (ii) a high level of TH1 cytokine production from PBMCs was induced by melanoma cells transduced with Adv-B7.1 plus Adv-IL12; (iii) the expression of HLA class I antigens, HLA class II antigens or ICAM-1 antigens was higher on melanoma cells transduced with Adv-lL12 or Adv-B7.1 plus IL12 than those transduced with Adv-LacZ or wild-type melanoma cells; and (iv) the expression of IL12 receptors on PBMCs was upregulated by melanoma cells transfected with Adv-IL12 or Adv-B7.1 plus IL12. Thus, melanoma cells transduced with both Adv-lL12 and B7.1 may represent another clinical approach for antimelanoma gene therapy.

Interstitial pneumonia complicated by Sjögren's syndrome, Hashimoto's disease, rheumatoid arthritis and primary biliary cirrhosis.

A 66-year-old woman diagnosed as having Hashimoto's disease and rheumatoid arthritis manifested interstitial pneumonia. We diagnosed Sjögren's syndrome and primary biliary cirrhosis as complications in this case. Steroid therapy was relatively effective for the interstitial pneumonia which was in an active state; however, during tapering of the steroid, there was a relapse and also severe dry throat. Cyclophosphamide was added and was effective in the prevention of recurrence. Even after discontinuation of steroid therapy, her general condition is stabilized. It is very important to carefully investigate other organ involvement as a prognostic factor in cases in which there are multiple autoimmune diseases.

Oncolytic adenovirus based on serotype 3.

Oncolytic adenoviruses have been safe in clinical trials but the efficacy has been mostly limited. All published trials have been performed with serotype 5 based viruses. The expression level of the Ad5 receptor CAR may be variable in advanced tumors. In contrast, the Ad3 receptor remains unclear, but is known to be abundantly expressed in most tumors. Therefore, we hypothesized that a fully serotype 3 oncolytic adenovirus might be useful for treating cancer. Patients exposed to adenoviruses develop high titers of serotype-specific neutralizing antibodies, which might compromise re-administration. Thus, having different serotype oncolytic viruses available might facilitate repeated dosing in humans. Ad3-hTERT-E1A is a fully serotype 3 oncolytic adenovirus controlled by the promoter of the catalytic domain of human telomerase. It was effective in vitro on cell lines representing seven major cancer types, although low toxicity was seen in non-malignant cells. In vivo, the virus had anti-tumor efficacy in three different animal models. Although in vitro oncolysis mediated by Ad3-hTERT-E1A and wild-type Ad3 occurred more slowly than with Ad5 or Ad5/3 (Ad3 fiber knob in Ad5) based viruses, in vivo the virus was at least as potent as controls. Anti-tumor efficacy was retained in presence of neutralizing anti-Ad5 antibodies whereas Ad5 based controls were blocked. In summary, we report generation of a non-Ad5 based oncolytic adenovirus, which might be useful for testing in cancer patients, especially in the context of high anti-Ad5 neutralizing antibodies.

Surface proteins and osteoblast markers: characterization of human adipose tissue-derived osteogenic cells.

INTRODUCTION: In adipose tissue-derived osteogenic cells (ADOC), osteoblast markers and surface proteins were determined and compared with osteoblasts harvested from cancellous bone (OB). METHOD: Osteocalcin (OC), core binding factor 1 (CBFA1), collagen type 1 (Coll1), alkaline phosphatase (ALP), nucleostemin (NS), and surface proteins CD 10, CD44, CD 59 and CD 105 were analyzed using RT-PCR, immunofluorescence and Western blot. RESULTS: Osteocalcin expression was more distinct in OB than in ADOC, but the other markers and surface proteins showed no differences. CONCLUSION: These data support the use of adipose tissue for future regenerative medicine; however, further studies are necessary to establish the role of long-term differentiation.

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

Reduced amplitude of the sural nerve sensory action potential in PARK2 patients.

The authors performed nerve conduction studies in nine PARK2 and eight idiopathic Parkinson disease patients and found a significant reduction of sural sensory nerve action potential (SNAP) amplitude in eight PARK2 patients who mostly remained asymptomatic. These data suggest that sensory axonal neuropathy may be a common clinical feature of PARK2 and a reduced amplitude of sural SNAP could be a diagnostic indicator of PARK2.

Functional characterization of a hybrid human-mouse interferon gamma receptor: evidence for species-specific interaction of the extracellular receptor domain with a putative signal transducer.

The human interferon gamma (IFN-gamma) receptor expressed in mouse cells displays binding properties indistinguishable from those of the resident receptor on human cells. Still, mouse cells expressing the human IFN-gamma receptor remain insensitive to human IFN-gamma. It is widely accepted that at least one species-specific cofactor encoded within human chromosome 21 is required for signal transduction. To define structural domains of the human IFN-gamma receptor responsible for this species-specific interaction, a hybrid between the human and the murine receptor was constructed and expressed in mouse L929 cells or in mouse L cell-derived SCC16-5 cells, which contain human chromosome 21. This hybrid receptor, which consisted of the extracellular domain of the human IFN-gamma receptor and the transmembrane and cytoplasmic domains of the murine IFN-gamma receptor, was found to bind human IFN-gamma with high affinity. However, only SCC16-5 cells expressing the human/mouse hybrid receptor were responsive to human IFN-gamma as revealed by enhanced expression of major histocompatibility complex class I antigens, induction of the transcription factor IRF-1, and induction of a partial antiviral state. These findings strongly suggest that IFN-gamma-mediated signal transduction requires a species-specific interaction of the extracellular portion of the known ligand-binding IFN-gamma receptor chain with an additional, presumably membrane-anchored receptor subunit.

Signaling steps involving the cytoplasmic domain of the interferon-gamma receptor alpha-subunit are not species-specific.

The ligand-binding chain of the interferon-gamma receptor (IFN-gamma R) is a unique cell surface protein which has no similarities to other cytokine receptors. Expression of this receptor chain (alpha-subunit) is not sufficient to mediate responsiveness to IFN-gamma. We and others have shown that IFN-gamma-mediated signal transduction requires a species-specific interaction of the extracellular portion of the known IFN-gamma receptor alpha-chain with an additional receptor subunit that was cloned recently and designated IFN-gamma R beta-chain or accessory factor 1. Here, we investigated whether this tight species barrier also applies to signaling events mediated by the cytoplasmic receptor domain. A cell line derived from embryos that lack the IFN-gamma R alpha-subunit was reconstituted with a hybrid mouse-human alpha-subunit that consisted of an extracellular murine and transmembrane and cytoplasmic human domains. The experiments reported herein showed that in mouse cells, the human intracellular domain of the hybrid IFN-gamma R alpha-subunit was fully functional and that, therefore, signaling steps involving this domain are not species-specific.

A novel attenuated replication-competent adenovirus for melanoma therapy.

To generate a replication-competent adenovirus (Ad) with specificity for melanoma, we constructed a tissue-specific promoter restricting E1A expression to melanoma cells. The combination of four copies of a mouse tyrosinase enhancer element (TE) fused to the human tyrosinase promoter (TP) yielded up to 2000-fold higher luciferase reporter activity in tyrosinase-expressing melanoma cells than in nonmelanoma cells. Insertion of the composite TETP construct upstream of the E1A gene was combined with deleting as far as possible the intertwined endogenous Ad enhancer/promoter (EP). The resulting AdDeltaEP-TETP vector, also deleted for the E3 region, was found to replicate in tyrosinase-positive melanoma cells, such as SK-Mel23 as efficiently as wild-type Ad5, but at a more than 50-fold reduced level in nonmelanoma tumour cells and primary human cells. Injection of AdDeltaEP-TETP into xenotransplanted melanomas, but not into HeLa-derived tumours led to long-lasting tumour regression in nude mice. This AdDeltaEP-TETP virus might be useful for the treatment of accessible lesions in advanced melanoma patients.

Studies of monoclonal antibodies specific for major histocompatibility complex products of the rat. IV. Production and characterization of polyclonal and monoclonal anti-anti-idiotypic antibodies.

Polyclonal syngeneic, allogeneic, and xenogeneic and monoclonal syngeneic anti-anti-idiotypic antibodies have been produced against previously described monoclonal anti-idiotypic antibodies with specificity for monoclonal RT1 alloantigen-specific antibodies. The anti-anti-idiotypes could again be shown to be highly specific for the monoclonal anti-idiotype used for the induction of the anti-anti-idiotypic antibodies and to carry the same, or a very similar, idiotype as the original monoclonal idiotypic antibody used to induce the monoclonal anti-idiotypic. Among the 30 syngeneic and allogeneic and the five xenogeneic polyclonal anti-anti-idiotypic antisera and the three monoclonal anti-anti-idiotypes, only one polyclonal antiserum showed binding capacity to the corresponding RT1-encoded antigenic determinants on spleen cells. All the other antibodies were idiotypic but not antigen binding.

Cloning of murine interferon gamma receptor cDNA: expression in human cells mediates high-affinity binding but is not sufficient to confer sensitivity to murine interferon gamma.

A full-length cDNA encoding the murine interferon gamma (IFN-gamma) receptor was isolated from a lambda gt11 library using a human IFN-gamma receptor cDNA probe. The deduced amino acid sequence of the murine IFN-gamma receptor shows approximately 53% homology to its human counterpart but no homology to other known proteins. Murine IFN-gamma receptor cDNA was expressed in human HEp-2 cells, which do not bind murine IFN-gamma and are insensitive to its action. Transfectants displayed the same binding properties as mouse cells. The biological responsiveness of such transfectants to various biological effects of both human and murine IFN-gamma was investigated, including modulation of major histocompatibility complex class I and class II antigen expression, inhibition of cell growth, and antiviral activity. Like parental HEp-2 cells, these transfectants responded only to human, but not to murine, IFN-gamma. Inversely, mouse L929 cells transfected with human IFN-gamma receptor cDNA were insensitive to human IFN-gamma. These results confirm and extend previous findings, suggesting that species-specific cofactors are needed for IFN-gamma-mediated signal transduction.