Mechanisms of in vivo anti-neuroblastoma activity of human natural IgM

Normal human sera of healthy adults contain natural IgM antibodies which are cytotoxic for human neuroblastoma cells. In this study, we evaluated the anti-neuroblastoma activity of these natural IgM antibodies in nude rats bearing solid human neuroblastoma tumours. A single intravenous (i.v.) injection of purified cytotoxic IgM led to uptake of IgM in the tumours with massive perivascular complement activation and accumulation of neutrophil granulocytes after 24 h. Five consecutive i.v. injections of purified cytotoxic IgM into neuroblastoma-bearing animals resulted in complete growth arrest of even large established solid tumours which lasted for several weeks after discontinuation of the injections, whereas tumours of control animals continued to grow exponentially during the observation period. These studies suggest that natural anti-neuroblastoma IgM may have a potential as a novel therapeutic modality in the treatment of human neuroblastoma.     

Rhabdomyolysis, acute renal failure and hepatopathy induced by lovastatin monotherapy

The immunoglobulin VH gene rearrangement in a primary cutaneous, large-cell (centroblastic and immunoblastic) B-cell lymphoma was analyzed using a micromanipulation/single-cell polymerase chain reaction technique. In all single B cells obtained from CD20-stained skin sections that gave a polymerase chain reaction product (eight of 27 in biopsy I), the same VHDJH rearrangement, consisting of DP-54-DIR1-JH3a genes, was detected, with no intraclonal nucleotide diversity. Comparison with the most closely related germline counterpart showed significantly altered complementarity determining gene regions as a result of somatic mutations, suggesting an antigen-driven selection and expansion ofthis particular B-cell clone. Interestingly, in a biopsy obtained from the patient 9 mo later, during disease progression (deep muscle infiltration), the lymphoma cells again contained the same VHDJH gene rearrangement (six of 18 in biopsy II) without any further somatic mutations. Therefore, it is suggested that the cutaneous lymphoma characterized throughout this study descended from postgerminal center B-cells.     

Skeletal muscle extra-aortic counterpulsation in dogs with dilated cardiomyopathy

Skeletal muscle extra-aortic counterpulsation was performed in seven dogs with dilated cardiomyopathy. A left latissimus dorsi dynamic descending thoracic aortomyoplasty was used as the autologous counterpulsator. Pulse train stimulation in diastole was used to initiate contraction and fibre type transformation. Two of the dogs died within 48 hours of surgery. The device was successfully activated in the five remaining dogs, but in one individual it failed within 48 hours of activation. Serial echocardiographic examinations of dogs in which the device functioned successfully (n = 4) showed trends towards the decrease in the left ventricular systolic internal dimension, left ventricular diastolic internal dimension, E-point to septal separation and left atrial diameter in systole seven to 14 days following the procedure, although these changes failed to persist in the long-term. The results suggest that skeletal muscle for cardiac assistances such as extra-aortic muscle counterpulsation, might be a therapeutic option for dogs with cardiac failure due to dilated cardiomyopathy.     

Rat renal allograft tolerance is associated with local TGF-beta and absence of IL-2r expression within chimeric immunocytic foci

Tolerance was induced in Lewis (LEW) rat renal allograft recipients of Brown Norway kidneys by multiple pretransplant donor-blood transfusions and prior limited cyclosporine A. Rat renal allograft tolerance was associated with the induction of systemic donor T cells (10%), an early phase of nonspecific suppressor-cell generation, followed by maturation of systemic antigen-specific suppressor cells, and renal cellular infiltrates that develop long-term in situ in the kidney graft model. It was hypothesized that these infiltrates represent chimeric immunocytic foci that are locally regulated via a TGF-beta-dependent mechanism. Both immunohistochemical staining and digital image analysis for cellular and extracellular TGF-beta, IL-2 receptor (CD25), and the BN Class I-MHC marker (OX-27) were performed. Control rejecting (REJ) kidneys did not demonstrate any differences with respect to levels of infiltrating immunocyte area vs long-term surviving (TOL) kidneys (3.9% vs 4.5%, P = .303). Immunostaining with the BN Class I MHC marker (OX-27) demonstrated high levels of chimerism within immunocyte foci of the tolerant grafts (OX-27 BN+immunocytes 49.0% +/- 5.1%). In situ cellular IL-2 receptor (CD25) expression was demonstrated in REJ kidney infiltrates but not within TOL immunocytic infiltrating foci, when measured as percent of total lymphocytes (REJ = 5.0% vs TOL = 0.4%, P = .031). Conversely, TGF-beta expression was significantly higher in immunocytes of TOL kidneys when measured as the number of DAB chromogen-staining pixels per total immunocyte area (TOL = .076 vs REJ = .047, P = .003). In conclusion, these results suggested that stable mixed immune chimerism (SMIC) plays an important role in DST-CyA-induced tolerance in situ. SMIC-induced tolerance may involve a local TGF-beta-dependent mechanism that is associated with in situ TGF-beta (+) and IL-2r (-) immunocytes.     

A molecular epidemiological probe for pig microchimerism

We have cloned and characterized a single-copy DNA sequence from the porcine alpha-1,3-galactosyltransferase gene that corresponds to a 547-base pair intron separating exons 3 and 4 of the protein coding domain. Polymerase chain reaction amplification of this sequence from flanking oligonucleotides generates a species-specific DNA probe (pgt34) capable of recognizing 50 pg chimeric template DNA at a pig to human cellular ratio of 1/10,000. Homologous DNA sequence is not identified in the macaque, baboon, or human genome by Southern hybridization. Analysis of a discordant model of pig to baboon xenotransplantation demonstrates peripheral blood microchimerism in the presence of a functioning pig kidney xenograft and persistence of microchimerism in lymphatic tissue after graft removal. This probe should be useful for tracking the fate of porcine cells in patients undergoing xenotransplantation of whole organs or free tissues such as pancreatic islet cells and should facilitate studies of microchimerism in experimental models of pig to monkey xenotransplantation.     

Activating Smoothened mutations in sporadic basal-cell carcinoma

Basal-cell carcinomas (BCCs) are the commonest human cancer. Insight into their genesis came from identification of mutations in the PATCHED gene (PTCH) in patients with the basal-cell nevus syndrome, a hereditary disease characterized by multiple BCCs and by developmental abnormalities. The binding of Sonic hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal inhibition by PTCH of Smoothened (SMO), a seven-span transmembrane protein. According to this model, the inhibition of SMO signalling is relieved following mutational inactivation of PTCH in basal-cell nevus syndrome. We report here the identification of activating somatic missense mutations in the SMO gene itself in sporadic BCCs from three patients. Mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Furthermore, skin abnormalities similar to BCCs developed in transgenic murine skin overexpressing mutant SMO. These findings support the role of SMO as a signalling component of the SHH-receptor complex and provide direct evidence that mutated SMO can function as an oncogene in BCCs.