Successful treatment of renal failure caused by multiple myeloma with HLA-identical living kidney and bone marrow transplantation: a case report.

Here we have described a successful HLA-identical living allogeneic kidney transplantation after bone marrow transplantation in a patient with end-stag liver disease caused by multiple myeloma (MM). Our case is unique, because this combined transplantation is rarely possible and because of our unique immunosuppressive and management strategies. A 45-year-old man with ESRD MM and κ light-chain nephropathy was diagnosed. Cytostatic treatment resulted in partial remission, so autologous peripheral stem cell transplantation (SCT) was performed leading to a complete remission; however the patient remained anuric. The patient's HLA-identical brother offered to be a donor of peripheral stem cells for collection and cryopreservation. Kidney transplantation was performed with a combination of tacrolimus sirolimuns, and methylprednisolone. With a well-functioning kidney graft, allogeneic SCT was performed in the incipient relapse phase of MM, after total body irradiation. Severe oropharyngeal infections, diarrhea, sepsis, and renal failure. Fearing acute renal rejection, we administered steroid bolus. He experienced therapy with gradual restoration of kidney function. Then, steroid-responsive acute graft-versus-host disease (grade II, predominantly bowel) was diagnosed on the background of diarrhea, which returned once. Later he experienced a left subclavian vein thrombosis at the site of a central venous catheter and sepsis. Having recovered from these events, the patient enjoys good health, with stable kidney function and normal protein excretion. After the steroid was stopped, a bone marrow biopsy revealed full-donor type normocellular hemopoiesis. Because of the chimerism, we gradually discontinued the immunosuppression including, sirolimus and finally tacrolimus, since with minimal trough levels there were no complications. Bone marrow biopsy showed a complete remission. In MM with ESRD HLA-identical combined kidney and bone marrow transplantation from a living donor may offer not only complete remission and good renal function, but also good health without immunosuppression.

Cochlear implants in five cases of auditory neuropathy: postoperative findings and progress.

OBJECTIVES: To review our experiences with some of the preoperative and postoperative findings in five children who were diagnosed with auditory neuropathy and were provided with cochlear implants. We describe changes in auditory function, which enabled these children to have significant improvement in their hearing and communication skills. STUDY DESIGN: Pre- and postoperatively, these children received complete medical examinations at Mayo Clinic, including related consultations in audiology, pediatrics, neurology, medical genetics, otolaryngology, psychology, speech pathology, and radiology. METHODS: These children typically had additional medical and audiological examinations at more than one medical center. The hearing assessments of these children included appropriate behavioral audiometric techniques, objective measures of middle ear function, acoustic reflex studies, transient (TOAE) or distortion product (DPOAE) otoacoustic emissions, auditory brainstem responses (ABR), and, in some cases, transtympanic electrocochleography (ECoG). After placement of the internal cochlear implant devices (Nucleus CI24), intraoperatively we measured electrode impedances, visually detected electrical stapedius reflexes (VESR) and neural response telemetry (NRT). These intraoperative objective measures were used to help program the speech processor for each child. Postoperatively, each child has had regular follow-up to assure complete healing of the surgical incision, to assess their general medical conditions, and for speech processor programming. Their hearing and communication skills have been assessed on a regular basis. Postoperatively, we have also repeated electrode impedance measurements, NRT measurements, otoacoustic emissions, and electrical auditory brainstem responses (EABR). We now have 1 year or more follow-up information on the five children. RESULTS: The five children implanted at Mayo Clinic Rochester have not had any postoperative medical or cochlear implant device complications. All of the children have shown significant improvements in their sound detection, speech perception abilities and communication skills. All of the children have shown evidence of good NRT results. All but case D (who was not tested) showed evidence of good postoperative EABR results. Otoacoustic emissions typically remained in the non-operated ear but, as expected, they are now absent in the operated ear. CONCLUSION: Our experiences with cochlear implantation for children diagnosed with auditory neuropathy have been very positive. The five children we have implanted have not had any complications postoperatively, and each child has shown improved listening and communication skills that have enabled each child to take advantage of different communication and educational options.

Ultra-trace analytical monitoring of silicon wafer surfaces by capillary electrophoresis.

Several methods are presented for the routine ultra-trace analytical monitoring of inorganic and organic anions and cations on the surface and in the native oxide of silicon wafers--the wafer-surface water-extraction method, the vapor-phase-decomposition method, and the re-dissolving method. Electrokinetic injection, sample stacking, and electrolyte composition were, therefore, optimized and made robust. For electrokinetic injection with transient isotachophoretic preconcentration a linear range of 0.05 to 0.5 micromol L(-1) was obtained; for sample stacking the linear range was 0.5 to 10 micromol L(-1), even in the presence of up to 750 micromol L(-1) hydrofluoric acid. Inorganic anions and monovalent carboxylic acids are predominately dissolved in the aqueous layer on the wafer surface whereas dicarboxylic acids are chemically bonded to the silanol groups and form esters.

Efficacy and safety studies of a recombinant chimeric respiratory syncytial virus FG glycoprotein vaccine in cotton rats.

Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.

Routine analysis of ultra pure water by ICP-MS in the low- and sub-ng/L level.

The chemical analysis with inductively coupled plasma-mass spectrometry (ICP-MS) can help to examine the purity of ultra pure water (UPW) down to 10 part per trillion (ng/L) and lower. For a proper determination of a high number of samples per week the analysis must be divided into two parts: the routine analysis and the reference water analysis. The routine analysis is done by direct measurement of the ultra pure water samples. Applying a standard addition method under particular clean conditions, the reference water analysis leads to the definition of the accurate zero. A quick evaluation scheme is also presented for the reference water analysis. The method is tested for its fitness for application by examining LOD (for relevant element < 2 ng/L), reproducibility and linearity of calibration. The ICP-MS was optimized according to the methodology of G. Taguchi to improve reproducibility and LOD.

Hearing and otopathology in Crouzon syndrome.

OBJECTIVES: To better establish the incidence and types of otologic and auditory abnormalities in patients with Crouzon syndrome. STUDY DESIGN: Retrospective chart review of the otologic and auditory findings of patients diagnosed with Crouzon syndrome who were seen at our institution between 1978 and 1994. METHODS: Charts were reviewed and data recorded on patient sex, family history, appearance, auricular abnormalities, auditory findings, history of otologic disease, and follow-up. RESULTS: Nineteen patients were identified with the diagnosis of Crouzon syndrome: 12 males and 7 females. Twelve cases represented spontaneous mutations. Eight patients had abnormalities involving the external ear: from malalignment of the pinna (6 patients) to external auditory canal atresia (1 patient). Ten patients had documented hearing loss: 4 with conductive hearing loss, 2 with a mixed hearing loss, and 4 with a sensorineural hearing loss, the etiologies of which ranged from ossicular fixation and serous otitis media to unknown sensorineural deficits. CONCLUSIONS: Patients with Crouzon syndrome can exhibit various pathological features of the ear. Although external malformations are unusual, middle ear disease and hearing loss are common. We advocate close otologic and audiologic follow-up in these patients and note a higher frequency of sensorineural hearing loss than previously reported. Recent genetic advances may allow more accurate and earlier diagnosis of this syndrome.

Total body irradiation before bone marrow transplantation. Technique and acute toxicity.

PURPOSE: To evaluate the total body irradiation methods in the National Institute of Oncology between January 1984 and February 1998. PATIENTS AND METHODS: One hundred and twenty-four patients underwent total body irradiation prior to bone marrow transplantation in the last 15 years. A special cobalt unit has been used, the dose rate was 6 to 8 cGy/min in the midline of the abdomen. The source-midline distance (SMD) was 340 cm and the field size was 80 x 200 cm. The dose calculation was done on the basis of a tissue-phantom ratio curve measured in total body irradiation conditions and effective tissue thickness (ETT). Between 1984 and 1992 the beam direction was horizontal, the patients laid in lateral position. In 11 cases the total dose to the abdominal midline was 10 Gy in 1 fraction. From 1986 the fractionation changed to 4 x 3 Gy in 4 days. Within individual lung shielding the average lung dose was 8.5 Gy. In 44/124 cases the order of conditioning treatment was chemo-radiotherapy. Since 1992 vertical beams were used, and the patients (80/124) laid in prone/supine position. The fractionation remained the same but radio-chemotherapy regime has been used. RESULTS: The irradiation in prone position proved to be safer than lateral because of smaller patient motion and it resulted in a more accurate positioning of lung shielding too. In all cases, the acute side effects (headache, nausea, vomiting) were moderate. Using radio-chemotherapy the acute side effects during the total body irradiation were uncommon and well tolerable. CONCLUSION: Our technique with the large source-midline distance, vertical beam direction and the supine/prone position is stable, convenient and safe to produce homogeneous dose distribution and ensures accurate and reproducible lung shielding.

Accurate calibration of TXRF using microdroplet samples.

TXRF has been applied in combination with VPD to the analysis of trace impurities in the native oxide layer of Si wafer surfaces down to the range of 10(8) atoms. cm(-2). Proper quantification of VPD/TXRF data requires calibration with microdroplet standard reference wafers. The precision of calibration function has been evaluated and found to allow quantification at a high level of 3 sigma confidence with microdroplet standard reference.

Fastin situ determination of the biomass of anchorage-dependent cells.

The lengthy and cumbersome protocol used to establish the growth kinetics characteristics of anchorage-dependent cells (ADC's)in situ (i.e. while the cells adhere on their microsupport) by Aperture Impedance Pulse Spectroscopy (AIPS) can be replaced by an accelerated procedure. This we have named Turbo AIPS whereby the same results can be obtained without actually performing the manipulations leading to the determination of the biomass.

HIV-1 envelope-elicited neutralizing antibody titres correlate with protection and virus load in chimpanzees.

In an attempt to compare the protective effect of vaccination with two forms of envelope antigens, and to define immunological correlates of protection against HIV infection, chimpanzees were vaccinated with either recombinant gp160 or gp120. Homologous HIV challenge was performed 3 weeks after the fourth immunization. The animal with the highest level of serum neutralizing antibodies (gp160 immunogen) was protected against HIV infection. All other chimpanzees became infected, but displayed various levels of infected PBMCs. The postchallenge data gave rise to the following conclusions: (1) protection correlated with the level of the serological immune response, but not with the nature of immunogen (gp120 versus gp160); (2) the virus-neutralizing titre at day of challenge correlated with protection from infection; (3) the relative magnitude of the lymphoproliferative T-cell response at day of challenge did not correlate with any protective effect; (4) the peak numbers of virus-infected PBMCs in vaccinated animals were lower than those observed in control animals, and this effect was correlated with the intensity of the antibody response at day of challenge. This raises the possibility that a beneficial effect of HIV vaccination may be achieved in a situation where sterile immunity is not consistently obtained.

Incomplete protection, but suppression of virus burden, elicited by subunit simian immunodeficiency virus vaccines.

We compared the efficacy of immunization with either simian immunodeficiency virus (SIV) Env glycoprotein (Env), Env plus Gag proteins (Gag-Env), or whole inactivated virus (WIV), with or without recombinant live vaccinia vector (VV) priming, in protecting 23 rhesus macaques (six vaccine and two control groups) from challenge with SIVmac251 clone BK28. Vaccination elicited high titers of syncytium-inhibiting and anti-Env (gp120/gp160) antibodies in all vaccinated macaques and anti-Gag (p27) antibodies in groups immunized with WIV or Gag-Env. Only WIV-immunized macaques developed anticell (HuT78) antibodies. After homologous low-dose intravenous virus challenge, we used frequency of virus isolation, provirus burden, and change in antibody titers to define four levels of resistance to SIV infection as follows. (i) No infection ("sterilizing" immunity) was induced only in WIV-immunized animals. (ii) Abortive infection (strong immunity) was defined when virus or provirus were detected early in the postchallenge period but not thereafter and no evidence of virus or provirus was detected in terminal tissues. This response was observed in two animals (one VV-Env and one Gag-Env). (iii) Suppression of infection (incomplete or partial immunity) described a gradient of virus suppression manifested by termination of viremia, declining postchallenge antibody titers, and low levels (composite mean = 9.1 copies per 10(6) cells) of provirus detectable in peripheral blood mononuclear cells or lymphoid tissues at termination (40 weeks postchallenge). This response occurred in the majority (8 of 12) of subunit-vaccinated animals. (iv) Active infection (no immunity) was characterized by persistent virus isolation from blood mononuclear cells, increasing viral antibody titers postchallenge, and high levels (composite mean = 198 copies per 10(6) cells) of provirus in terminal tissues and blood. Active infection developed in all controls and two of three VV-Gag-Env-immunized animals. The results of this study restate the protective effect of inactivated whole virus vaccines produced in heterologous cells but more importantly demonstrate that a gradient of suppression of challenge virus growth, reflecting partial resistance to SIV infection, is induced by subunit vaccination. The latter finding may be pertinent to studies with human immunodeficiency virus vaccines, in which it is plausible that vaccination may elicit significant suppression of virus infection and pathogenicity rather than sterilizing immunity.

Dielectric spectroscopy of mammalian cells. 2. Simultaneous in situ evaluation by aperture impedance pulse spectroscopy and low frequency dielectric spectroscopy of the biomass of HTC cells on Cytodex 3.

Low-frequency dielectric spectroscopy has been used in situ, i.e. while the cells are still attached to their microsupport, to monitor the changes of biomass accompanying the growth of anchorage-dependent cells. This method, when compared to Aperture Impedance Pulse Spectroscopy (also called electronic sizing), is characterized by a somewhat lower degree of resolution. Suggestions are made on how to determine the capacitance of the spent growth medium alone, still keeping the probe inserted in the bioreactor. This will make dielectric spectroscopy the first truly in situ, on-line, in real time, non-invasive measure of the biomass.

Dielectric spectroscopy of mammalian cells. 1. Evaluation of the biomass of HeLa- and CHO cells in suspension by low-frequency dielectric spectroscopy.

The capacitance of suspensions of CHO and HeLa cells (0.5-3 x 10(6) cells/ml) has been measured between 0.2 and 10 MHz. As frequencies decrease, there is a continuous increase in capacitance of both the cell suspension and the spent growth medium free of cells, a phenomenon which is partially attributed to an increased polarisation of the electrodes. At a given frequency, subtraction of the capacitance of the spent medium from that of the cell suspension allows one to determine the capacitance of the cells only. The intensity of this signal varies linearly with the biomass and cell size. At low frequencies such as those used in this study (0.25 MHz), where sensitivity is the highest, concentrations as low as 0.5 x 10(6) cells/ml can be accurately measured. Suggestions are made how to make these measures on-line, non-invasive and in real time.

Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines.

The cDNA encoding human myeloperoxidase carries three ATG codons in frame; 144, 111 and 66 bp upstream from the proprotein DNA sequence. In order to determine the most efficient signal sequence, three cDNA modules starting at each of the ATG were cloned into an eucaryotic expression vector and stably expressed in Chinese hamster ovary cell lines. In all three cases, recombinant human myeloperoxidase (recMPO) was secreted into the culture medium of transfected cells, indicating that each of the signal peptides functions efficiently. One of the recombinant cell lines, which was amplified using methotrexate, overexpresses enzymatically active recMPO up to 6 micrograms.ml-1.day-1. The recombinant product was purified by a combination of ion-exchange and metal-chelate chromatography, and characterized in terms of molecular mass, amino-terminal amino acid analysis, glycosylation, physicochemical properties and biological activity. The data show that recMPO is secreted essentially as a monomeric, heme-containing, single-chain precursor of 84 kDa which exhibits peroxidase activity. Amino-terminal analysis indicated that cleavage of the signal peptide occurs between amino acids 48 and 49. In addition, recMPO appeared to be glycosylated up to the last stage of sialylation, to an extent similar to that of the natural enzyme. Specific activity measurements as well as stability data, in various pH, temperature, ionic strength and reducing conditions, indicated that the recombinant single-chain enzyme behaves essentially in the same way as the natural two-chain molecule. Finally, recMPO was shown to exert potent cytotoxicity towards Escherichia coli when provided with its physiological substrates, i.e. hydrogen peroxide and chloride ions.

Chromosome aberrations in mixed cultures of in vitro irradiated and unirradiated human lymphocytes.

Human whole blood samples were exposed to different doses (1, 2, 4, 6 or 10 Gy) of gamma-radiation and mixed with different volumes of non-irradiated blood before culturing to simulate partial body irradiations. Chromosome aberrations were analysed and the frequency of dicentrics was found to be lower than expected, particularly when irradiated blood was mixed with large volumes of non-irradiated blood and after exposure to high radiation doses. For the mixtures of irradiated and unirradiated blood the deviation from the Poisson distribution depends on the respective proportions and on the doses. The results can be correlated to in vivo aberration frequencies in case of therapeutical treatments, but the yields of aberrations are generally underestimated in vitro.

Shuttle transfer (or retrotransfer) of chromosomal markers mediated by plasmid pULB113.

The IncP1 plasmid pULB113 (RP4::miniMu) not only mediates the transfer of chromosomal markers in the classical direction (i.e. from the donor to the recipient cell) but also in the opposite direction (i.e. from the recipient bacterium to the donor). This phenomenon of retrotransfer was observed in homologous matings with Pseudomonas fluorescens, Alcaligenes eutrophus and Salmonella typhimurium. Retrotransconjugants could be discriminated from direct transconjugants by appropriate chromosomal and plasmid markers used to distinguish the mating partners not bearing pULB113. Retrotransfer of chromosomal markers occurred at frequencies equal to, or sometimes greater than, those observed for the direct mobilization, thus allowing the recovery of "recipient" recessive markers in the "donor" with linkage values similar to those found in the normal direction. Retrotransfer was also observed in heterospecific matings involving A. eutrophus and pULB113 bearing P. fluorescens: R-primes carrying different selected and unselected markers were recovered in both bacteria. "Retrotransfer" or "shuttle transfer" seems to be a specific trait of IncP1 plasmids.

Cytogenetic damage induced in human lymphocytes by low doses of 60Co gamma rays delivered at high and low dose rates.

Chromosomal aberrations were investigated in human peripheral blood lymphocytes after exposure to low doses of 60Co gamma rays delivered acutely or at low dose rates (0.1 or 0.03 Gy/h). Chromosome analysis was performed in cells collected after a 44 to 46 hour culture time in order to avoid scoring of second division cells, and the dose-related induction of aberrations was analysed by the maximum likelihood method. In all cases, the induction of dicentrics was well described by a linear-quadratic dose response model (Y = aD + bD2), the data obtained at a low dose rate being equally well fitted to a linear equation. According to earlier findings on the mechanisms of aberration formation, the two lesions originating from single ionizing tracks have to be produced within a period of approximately 5 hours, in order to interact and to give rise to dicentric aberrations, which could explain the decrease in the quadratic term at the low dose rate since the highest doses were delivered over a period of more than 5 hours.