Unusual Cystic Fibrosis Transmembrane Conductance Regulator Mutations and Liver Disease: A Case Series and Review of the Literature.

Cystic fibrosis (CF) is caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene, deranging the activity of chloride channels on the epithelial cell surface. Herein we describe end-stage liver disease in 3 infants with rare CFTR gene mutations; 2 of them were heterozygous. Case 1 was a premature male infant with negative CF screening at birth who developed a small bowel obstruction in the neonatal period requiring an ileostomy, with subsequent cholestatic liver disease and portal hypertension. In addition, he was noted to have frequent respiratory infections prompting a sweat test, which was positive. Genetic testing revealed that he was heterozygous for P.1177F. He then underwent a successful liver transplant. Case 2 was a female infant who developed progressive cholestasis with poor weight gain and was found to have neonatal hepatitis on liver biopsy. A sweat test was negative and genetic testing revealed she was heterozygous for CFTR and PEX26 gene mutations. She subsequently developed pneumatosis involving the cecum that was treated conservatively, followed by a successful liver transplant. Case 3 was a male infant who developed progressive liver disease, with liver biopsy showing neonatal hepatitis. He was extensively investigated but had a negative sweat test on repeated studies. Genetic testing revealed that the patient was heterozygous P.K186N-variant in the AKRID1 gene and homozygous P.R75Q-variant in the CFTR gene. Unfortunately, he succumbed to an acute upper gastrointestinal hemorrhage. Rare and unusual CFTR mutations, even in the heterozygous form, may be a feature in otherwise undiagnosed end-stage liver disease of infancy.

Immunogenicity of a prophylactic quadrivalent human papillomavirus L1 virus-like particle vaccine in male and female adolescent transplant recipients.

Organ TX recipients are at an increased risk of developing cancers of the lower genital tract related to HPV. The quadrivalent HPV vaccine has high efficacy in preventing these diseases, but response to many vaccines is suboptimal after organ transplantation. Liver and kidney TX recipients received quadrivalent HPV vaccine. Serum samples were tested for anti-HPV levels. Of 20 renal transplant recipients screened, 14 received vaccine. Of these, seven completed the vaccine series and seven had incomplete vaccination. Of five liver TX children, three received vaccines (two complete and one incomplete). All eight kidney and liver TX children with complete vaccination and available results were seronegative at baseline and had seroconversion at month 7 for all four HPV types. Six of 14 (42.8%) kidney TX recipients developed AR. During the same time period, eight of 28 (28.5%) non-vaccine renal transplant recipients developed AR (p = ns). Transplant adolescents developed 100% seroconversion to all four HPV serotypes with HPV vaccine with serologic titers similar to historic controls. A non-significant increased incidence of AR was noted among kidney transplant vaccine recipients. A much larger study would be needed to evaluate whether HPV vaccination increases AR in transplant adolescents.

Clinicopathologic features of post-transplant lymphoproliferative disorders arising after pediatric small bowel transplant.

Few studies examined the clinicopathologic features of PTLD arising in pediatric SBT patients. Particularly, the association between ATG and PTLD in this population has not been described. Retrospective review of 81 pediatric patient charts with SBT--isolated or in combination with other organs--showed a PTLD incidence of 11%, occurring more frequently in females (median age of four yr) and with clinically advanced disease. Monomorphic PTLD was the most common histological subtype. There was a significant difference in the use of ATG between patients who developed PTLD and those who did not (p < 0.01); a similar difference was seen with the use of sirolimus (p < 0.001). These results suggested a link between the combination of ATG and sirolimus and development of more clinically and histologically advanced PTLD; however, the risk of ATG by itself was not clear. EBV viral loads were higher in patients with PTLD, and median time between detection of EBV to PTLD diagnosis was three months. However, viral loads at the time of PTLD diagnosis were most often lower than at EBV detection, thereby raising questions on the correlation between decreasing viral genomes and risk of PTLD.

Abnormal CX3CR1⁺ lamina propria myeloid cells from intestinal transplant recipients with NOD2 mutations.

Although progress has been made in intestinal transplantation, chronic inflammation remains a challenge. We have reported that the risk of immunological graft loss is almost 100-fold greater in recipients who carry any of the prevalent NOD2 polymorphisms associated with Crohn's disease, and have shown that the normal levels of a key antimicrobial peptide produced by the Paneth cells of the allograft, fall as the graft becomes repopulated by hematopoietic cells of the NOD2 mutant recipient. These studies are extended in this report. Within several months following engraftment into a NOD2 mutant recipient the allograft loses its capacity to prevent adherence of lumenal microbes. Despite the significantly increased expression of CX3CL1, a stress protein produced by the injured enterocyte, NOD2 mutant CX3CR1(+) myeloid cells within the lamina propria fail to exhibit the characteristic morphological phenotype, and fail to express key genes required expressed by NOD2 wild-type cells, including Wnt 5a. We propose that the CX3CR1(+) myeloid cell within the lamina propria supports normal Paneth cell function through expression of Wnt 5a, and that this function is impaired in the setting of intestinal transplantation into a NOD2 mutant recipient. The therapeutic value of Wnt 5a administration in this setting is proposed.

Anti-tumor activity of motesanib in a medullary thyroid cancer model.

BACKGROUND: Medullary thyroid cancer (MTC) is frequently associated with mutations in the tyrosine kinase Ret and with increased expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Motesanib is an investigational, orally administered small molecule antagonist of VEGFR1, 2, and 3; platelet-derived growth factor receptor (PDGFR); Kit; and possibly Ret. AIM: The aim of this study was to investigate the effects of motesanib on wildtype and mutant Ret activity in vitro and on tumor xenograft growth in a mouse model of MTC. METHODS/RESULTS: In cellular phosphorylation assays, motesanib inhibited the activity of wild-type Ret (IC(50)=66 nM), while it had limited activity against mutant Ret C634W (IC(50)=1100 nM) or Ret M918T (IC(50)>2500 nM). In vivo, motesanib significantly inhibited the growth of TT tumor cell xenografts (expressing Ret C634W) and significantly reduced tumor blood vessel area and tumor cell proliferation, compared with control. Treatment with motesanib resulted in substantial inhibition of Ret tyrosine phosphorylation in TT xenografts and, at comparable doses, in equivalent inhibition of VEGFR2 phosphorylation in both TT xenografts and in mouse lung tissue. CONCLUSIONS: The results of this study demonstrate that motesanib inhibited thyroid tumor xenograft growth predominantly through inhibition of angiogenesis and possibly via a direct inhibition of VEGFR2 and Ret expressed on tumor cells. These data suggest that targeting angiogenesis pathways and specifically the VEGF pathway may represent a novel therapeutic approach in the treatment of MTC.

Complex arterial reconstruction in multivisceral transplantation.

We report the case of a successful multivisceral transplant in which both donor and recipient presented aberrant anatomy of the celiac-mesenteric axis requiring five separate arterial anastomoses to reconstruct the blood inflow to the graft.

Rejection reversibly alters enteroendocrine cell renewal in the transplanted small intestine.

Acute small intestinal allograft rejection presents clinically as an abrupt increase in ileal fluid output in the absence of extensive inflammation. We questioned whether acute intestinal rejection might be accompanied by a disturbance of normal intestinal stem cell differentiation. We examined the intestinal epithelial secretory cell lineage among patients experiencing early rejection before and during rejection as well as following corrective therapy. Lineage-specific progenitors were identified by their expression of stage-specific transcription factors. Progenitors of the enteroendocrine cell (EEC) expressing neurogenin-3 (NEUROG3) were found to be disproportionately reduced in numbers, along with their more mature EEC derivatives expressing neuro D; the enteric hormone PYY was the most profoundly depleted of all the EEC products evaluated. No change in the numbers of goblet or Paneth cells was observed. Steroid treatment resulted in resolution of clinical symptoms, restoration of normal patterns of EEC differentiation and recovery of normal levels of enteric hormones. Acute intestinal rejection is associated with a loss of certain subtypes of EEC, most profoundly, those expressing PYY. Deficiency of the mature EECs appears to occur as a consequence of a mechanism that depletes NEUROG3 EEC progenitors. Our study highlights the dynamics of the EEC lineage during acute intestinal rejection.

NOD2-expressing bone marrow-derived cells appear to regulate epithelial innate immunity of the transplanted human small intestine.

BACKGROUND: Intestinal allograft rejection resembles Crohn's disease clinically and pathologically. An understanding of its mechanism could impact this life-saving procedure, as well as provide insight into the pathophysiology of inflammatory bowel disease. The NOD2 protein has been implicated as a key player in intestinal immune health, as a consequence of the discovery of three polymorphisms linked with Crohn's disease. An investigation was carried out to determine whether epithelial immune function and graft survival were influenced by NOD2 mutations in an intestinal transplant population. METHODS: The NOD2 genotypes of 34 transplants performed consecutively over the past 3 years were determined. The NOD2 genotypes were related to clinical outcomes and the expression of certain intestinal antimicrobial peptides (AMPs) believed to protect the epithelium. RESULTS: An unexpectedly high percentage of recipients, 35%, possessed NOD2 polymorphisms, while 8.6% of donors had comparable mutations. The likelihood of allograft failure was about 100-fold higher in recipients with mutant NOD2 alleles compared with recipients with wild-type NOD2 loci. Rejection in NOD2 mutant recipients was characterised by decreased expression of certain Paneth cell and enterocyte AMPs, prior to the onset of epithelial injury and inflammation. CONCLUSIONS: Crohn's disease-associated polymorphisms in the NOD2 gene in the recipient represent a critical immunological risk factor for intestinal allograft rejection. Compromised epithelial defences precede visible epithelial injury and inflammatory infiltration. The association of impaired epithelial immunity with the recipient's genotype suggests that certain NOD2-expressing cells of haematopoietic origin play a role in the process, perhaps by regulating expression of certain epithelial AMPs within the allograft.

HCV genotype distribution among HIV co-infected individuals in Argentina: Relationship with host and viral factors / Distribución de los genotipos de HCV entre individuos co-infectados con HIV en Argentina: su relación con factores del huésped y del virus

Coinfection with hepatitis C virus (HCV) in individuals infected with HIV is associated with a higher incidence of liver injury, hepatic decompensation, anddecreased survival than that observed in an HIVmonoinfected population. While prevalence studies on HIV/HCV coinfection have been performed in theU.S. and in some European countries, little is known about HCV genotype distribution in Latin America.The main objective was to evaluate the HCV prevalence and genotypes among HIV co-infected patients, and their relationship with HCV viral load, serumALT level and T lymphocyte CD4+ cell count. These data pursue to increase the knowledge from South America about a pressing problem from HIV-infectedpatients. Retrospectively collected specimens from 593 HIV-positive individuals in Argentina were tested foranti-HCV. These were analyzed for HCV-RNA qualitatively and quantitatively. The HCV genotype was determined by the RFLP method. One hundred andtwenty-nine (21.7%) HIV-infected individuals were anti-HCV positive; 65.9% of them exhibited detectable HCV- RNA. Genotype 1 (43, 1a/c; 9, 1b;and 5, 1a/c+1b) was present in 57, while 1, 14 and 13 were infected with genotype 2, 3 or a mix, respectively.Co-infected individuals were more likely to be male, without significant differences in age and CD4+ cell counts than HIV-monoinfected individuals.HCV infection prevalence in patients co-infected with HIV highlights the impending public health impact of this problem. Considering the increasingrate of HCV genotypes with lower response rates to treatment among HIV co-infected patients, antiretroviraltherapy success might be jeopardized by HCV coinfection.

La coinfección con el virus de hepatitis C (HCV) en individuos infectados con HIV está asociada con una mayor incidencia de injuria y descompensación hepática,y un menor tiempo de supervivencia respecto de la población mono-infectada por HIV. Mientras que diferentesestudios de prevalencia de la coinfecciónHIV/HCV se han llevado a cabo en Estados Unidos y países de Europa, la información de la distribución degenotipos de HCV en Latinoamérica es escasa. El objetivo de este estudio fue evaluar la prevalencia de HCVy la distribución de sus genotipos entre pacientes coinfectados con HIV, y su relación con la carga viral deHCV, los niveles séricos de ALT y el recuento de linfocitos T CD4+. Estos datos pretenden incrementar el conocimiento desde la región de Sudamérica acerca de este acuciante problema en pacientes infectados con HIV.Retrospectivamente se colectaron especímenes desde 593 pacientes infectados con HIV en Argentina enquienes se investigó la presencia de anticuerpos anti-HCV. Se pesquisó además la presencia de RNA viral deHCV tanto cualitativa como cuantitativamente. El genotipo de HCV se determinó por la técnica de RFLP.Ciento veintinueve (21.7%) individuos infectados con HIV fueron positivos para anti-HCV; 65.9% de ellos exhibieron RNA de HCV detectable. El genotipo 1(43, 1a/c; 9, 1b; y 5, 1a/c+1b) se presentó en 57 individuos, en tanto que 1, 14 y 13 estaban infectados porlos genotipos 2, 3 o mezcla de ellos, respectivamente. Predominó el sexo masculino entre los individuos concoinfección, en tanto que no se advirtieron diferencias significativas respecto de los pacientes infectados sólocon HIV en lo referido a edad y recuento de linfocitos T CD4+. La prevalencia de infección por HCV en pacientescoinfectados con HIV resalta el impacto de esta problemática en la salud pública. Considerando la creciente tasa de genotipos de HCV con menor respuestaal tratamiento entre los pacientes coinfectados con HIV, el efecto...

Candidiasis esofágica: análisis clínico y micológico / Oesophageal candidiasis: clinical and mycological analysis

Oesophageal candidiasis is an epithelial infection which requires an immune deficiency. C. albicans is commonly the cause, although other species may also be responsible. Resistance to fluconazole, drug of choice for treatment, is an emerging problem. The objectives of the current paper were: to determine the frequency of oesophageal candidiasis in patients submitted to upper gastrointestinal endoscopy, analyze risk factors, identify Candida species and determine in vitro susceptibility to fluconazole. During 12 months, 34 patients with oesophageal candidiasis were detected. Out of 1.230 HIV negative and 91 HIV positive patients submitted to upper endoscopy, 11 (0.9%) and 23 (25.3%), respectively, had candidiasis. Risk factors for HIV negative patients were systemic antibiotic therapy in 2, deficient dental cleaning in 2 aged patients, use of proton pump inhibitors in 3, inhaled steroids in 2, malignancy in 1 and oral steroids in 1. The histopathologic diagnosis was confirmed in 48.6% of cases. Cultures were positive in 91.2% C. albicans was prevalent (93.5%), and was associated to other species in 5 cases (16.1%), (3 C. glabrata, 1 C. tropicalis and 1 C. parapsilosis). One case cultured only C. glabrata and 1, only C tropicalis. Out of 31 cultures, 25 were susceptible to fluconazole, 4 dose dependent (1 C. albicans, 3 C. glabrata), and 2 resistant (1 C. albicans, 1 C. glabrata). Frequency of oesophageal candidiasis was low, except for HIV positive patients. The most common etiologic agent was C. albicans, though other Candida species were also found. C. albicans and C. glabrata showed dose dependency and resistance to fluconazole.

La candidiasis esofágica es una infección epitelial querequiere un defecto adicional inmunitario. Candida albicans es la especie más frecuente, aunque puedenencontrarse otras. Un problema emergente es la resistenciaal fluconazol, droga de elección para tratarla. Los objetivos fueron: determinar la frecuencia de candidiasisesofágica en pacientes sometidos a endoscopía, analizar los factores predisponentes, identificar las especiescausantes, y estudiar la sensibilidad in vitro al fluconazol. Durante 12 meses se realizaron 1.321 endoscopíasdonde se detectaron 34 pacientes con candidiasis esofágica. Se hicieron 1.230 endoscopías en pacientes HIV negativos y 91 en HIV positivos. Se diagnosticó candidiasis esofágica en 11 (0.9%) y 23(25.3%), respectivamente. En HIV negativos, fueron causas predisponentes: antibioticoterapia prolongada, prótesis dentarias sin higiene, uso prolongado de inhibidoresde la bomba de protones, secreción ácida, corticoides inhalatorios, malignidad y vasculitis bajo corticoterapia. La histopatología fue positiva en 48.6%. El cultivo se desarrolló en el 91.2%. C. albicans fue laespecie más frecuente (93.5%) y en 5 pacientes (16.1%) se la encontró asociada a C. glabrata (3) C. tropicalis (1) y C. parapsilosis (1). En un caso solo se cultivó C. glabrata y en otro C. tropicalis. De las 31 cepas, 25 fueron sensibles al fluconazol, 4 dosis dependientes (1 C. albicans, 3 C. glabrata), y 2 resistentes(1 C. albicans, 1 C. glabrata). En nuestro hospital, la frecuencia de candidiasis esofágica fue baja, excepto enHIV positivos. El principal agente etiológico fue C. alalbicans,aunque también se cultivaron otras especies. C. albicans y C. glabrata mostraron dosis dependencia yresistencia al fluconazol.

Erythromycin-resistant Streptococcus pyogenes in Argentina

Erythromycin (ERY) resistance in Streptococcus pyogenes has recently emerged as a problem of growing concern all through the world. We are presenting the comparison of results of the continuous surveillance of erythromycin resistance in S. pyogenes performed since 1989 in the Hospital de Pediatría J.P.Garrahan of Buenos Aires City, with independently observed rates in other five centers of Buenos Aires and seven centers of six other Argentinian cities, obtained between 1999 and 2001. A significant increase of erythromycin resistance was observed among S. pyogenes isolated in the Hospital Garrahan (6.6% in 1998-1999 to 9.9% in 2000). Similar trends were also detected in other centers of other Argentinian cities when recent data were compared to results of a multicenter study performed in 1995. However, lower rates of resistance were recorded in Mendoza, Cipolletti and Neuquén in comparison with data of 1995, 1998 and 1998 respectively. The reason of such decreasing resistance rates deserves to be investigated. The average of ERY-resistance rates obtained in the surveyed centers was 6.7% (range 0.5-14.1%). Control of antimicrobial use should be performed to warrant the future effectiveness of macrolide antibiotics regarding the positive association between use and resistance. These results also suggest that susceptibility tests for macrolides should be performed whenever S. pyogenes is isolated in Argentina.

La resistencia a la eritromicina en Streptococcus pyogenes ha emergido en los últimos tiempos como un problema creciente en todo el mundo. En este trabajo se presenta la comparación de los resultados de la vigilancia continua de la resistencia a la eritromicina que se viene realizando en el Hospital de Pediatría J.P.Garrahan de Buenos Aires desde 1989, con resultados independientes de otros cinco centros de la ciudad de Buenos Aires y siete de otras seis ciudades argentinas, obtenidos entre 1999 y 2001. Se observó un aumento significativo en el Hospital Garrahan (6.6% en1998-1999 a 9.9% en el año 2000) y una tendencia similar en otros centros de diversas ciudades argentinas si secomparan estos datos con los de un estudio multicéntrico realizado en 1995. No obstante, se registraron menoresporcentajes de resistencia en Mendoza, Neuquén y Cipolletti, en relación a lo hallado en 1995, 1998 y 1998respectivamente. La razón de esta disminución merece ser investigada. El porcentaje promedio de resistencia aeritromicina obtenido en los distintos centros participantes de este estudio fue de 6.7% (rango 0.5-14.1%). Debeefectuarse un control en el uso de estos antibióticos para garantizar la efectividad futura de los macrólidos, teniendo en cuenta la asociación estrecha entre uso y resistencia. Estos resultados sugieren que deberían realizarse pruebas de sensibilidad a los macrólidos para todos los aislamientos de S. pyogenes en la Argentina.

Comparación de métodos para la identificación de las levaduras más frecuentes en el laboratorio de microbiología clínica / Routine identification of commonly isolated yeast in clinical microbiology

Se evaluaron diferentes métodos para la identificación de rutina de levaduras de importancia médica. Se estudiaron 150 aislamientos provenientes de muestras clínicas: 25 C.albicans, 25 C.tropicales, 25 C.glabrata, 25 C.parapsilosis, 8 C.guiliermondii, 11 C.krusei y 31 Cryptococcus neoformans, con las tarjetas Yeast Biochemical Card bioMerieux Vitek (YBC), CHROMagar Candida (CHR) y la asociación de ambas técnicas con el Agar Morfología (AM). El método de referencia fue API 20C, formación de tubo germinativo, AM, agar urea Christensen y agar semilla girasol. YBC identificó 135 (90 por ciento) levaduras. La sensibilidad (S) y especificidad (E) fue: 92-98 por ciento para C.albicans y C.tropicalis; 84-99 por ciento para C.parapsilosis; 100-99 por ciento para C.glabrata; 91-100 por ciento para C.krusei; 63-98 por ciento para C.guilliermondii y 90-99 por ciento para Cryptococcus neoformans, respectivamente. CHR identificó correctamente el 100 por ciento de C.albicans, el 92 por ciento de C.tropicalis y el 91 por ciento de C.krusei. Ambos métodos combinados con AM alcanzan un 100 por ciento de S.y E. YBC fue apropiado para la identificación de levaduras aisladas de muestras clínicas. CHR fue efectivo y fácil de usar para la identificación de C.albicans, C.tropicalis y C.krusei. Se recomienda utilizar el AM con ambos métodos.

Falsos cultivos positivos por contaminación cruzada en laboratorios de tuberculosis / False-positive cultures due to cross contamination in tuberculosis laboratories

Fifteen episodes of Mycobacterium tuberculosis laboratory cross-contamination suspected between 1996 and 2001 at 6 laboratories in Buenos Aires City and suburbs were investigated by IS6110 RFLP. Thirteen episodes were confirmed. Even though BACTEC 460 produced the highest number of confirmed episodes in a single laboratory, the most extended one occurred while employing conventional culture procedures in solid medium. The double repetitive element-polymerase chain reaction (DRE-PCR) was applied to 8 of these episodes and produced concordant results with those of the RFLP. The DRE-PCR appears to be a valuable tool for the prompt identification of false positive cultures. The timely rectification of defects in laboratory protocols can avert false diagnoses of tuberculosis and unnecessary prolonged treatments.

The effects of keratinocyte growth factor in preclinical models of mucositis.

The epithelium of the oral cavity and small intestine of the gastrointestinal tract have a high rate of cell renewal and as such, are sensitive to cytotoxic therapies that kill rapidly dividing cells. Mucositis is a complication of cancer therapy where impairment of the regenerative capacity of the epithelium leads to atrophy, ulceration and a loss of barrier function. Keratinocyte growth factor (KGF) is an epithelial cell-specific growth and differentiation factor that is trophic for the mucosal epithelium of the gastrointestinal tract. In this study, KGF in normal animals caused epithelial thickening in the squamous epithelium of the oral cavity and increased crypt depth and villus height of the small intestine. It also appeared to regulate gene expression in these tissues including that of some antioxidant enzymes and intestinal trefoil protein. KGF has been shown to be efficacious in several preclinical models of mucositis where KGF pretreatment reduced weight loss typically seen during and after the course of therapy and significantly improved survival. At a tissue level KGF reduced atrophy, accelerated regrowth, and decreased ulcer formation of the oral epithelium after irradiation, and improved crypt survival and prevented villus atrophy in the small intestine of irradiated or chemotherapy-treated mice. Preliminary studies suggest that its efficacy may be partly a consequence of the growth and differentiation effect, and also partly due to regulation of the expression of genes that play a role in mucosal protection. These data suggest that KGF may be useful for the prevention or treatment of mucositis in patients treated with regimens of cancer therapy that have gastrointestinal toxicity.

Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses.

This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.

Influence of a short or long abstinence period on semen parameters in the ejaculate of patients with nonobstructive azoospermia.

OBJECTIVE: To compare the effect of a short (4 days) or a long (14 days) abstinence period on sperm retrieval by extended sperm preparation in patients with nonobstructive azoospermia scheduled for testicular biopsy and intracytoplasmic sperm injection (ICSI). DESIGN: A prospective case control study. SETTING: Male infertility clinic in a university hospital. PATIENT(S): Fifty male patients with nonobstructive azoospermia, scheduled for testicular biopsy for ICSI. INTERVENTION(S): Diagnosis of nonobstructive azoospermia and a thorough microscopic search for sperm cells (extended sperm preparation). MAIN OUTCOME MEASURE(S): The number of sperm cells collected, sperm motility, and total motile sperm count after short and long abstinence periods. RESULT(S): There was a significant difference between long and short abstinence with an increase in sperm count (log-to-log transformed analysis of variance P<.025) and total motile sperm (P<.025 analysis of variance, P<.02 paired Student's t-test) in the former group, but no significant change in sperm motility (Wilcoxon and paired Student's t-test). In 18 patients, sperm concentration and sperm motility were similar in a second collection, done after the same abstinence period, compared with the same parameters in the first sample. When at least 10 motile sperm were defined as the cutoff number, allowing ICSI without testicular biopsy, no significant differences were found between the two abstinence periods. No clinical or laboratory male characteristic could predict the detection of 10 motile sperm by extended sperm preparation either after a short or a long abstinence period. CONCLUSION(S): Sperm count and total motile sperm were increased after a long abstinence period, with no change in sperm motility. No additional advantages were conferred by long abstinence as opposed to short abstinence when 10 motile sperm were defined as the cutoff number for ICSI. The recommended period of abstinence for extended sperm preparation and ICSI, whether short or long, should be individualized for each patient.

Adrenomedullin increases fluid extravasation from the splenic circulation of the rat.

1. We studied the effect of adrenomedullin (ADM) on fluid efflux from the splenic vasculature into extravascular spaces. 2. Splenic arterial infusion of ADM (1, 3 and 9 ng min(-1); n = 9, 11 and 10, respectively) caused a dose-dependent increase in intrasplenic fluid efflux (+0.6 +/- 0.3 (saline) vs. +2.0 +/- 0.3 ml min(-1) (9 ng min(-1) ADM), P < 0.05), and in splenic (venous minus arterial) haematocrit (+0.8 +/- 0.1 (saline, n = 6) vs. +3.1 +/- 0.3 % (9 ng min(-1) ADM, n = 7), P < 0.05). There was no change in splenic weight (0.99 +/- 0.02 (saline, n = 6) vs. 0.99 +/- 0.02 g (9 ng min(-1) ADM, n = 7), P > 0.05). 3. There was no change in MAP before (97.5 +/- 2.2 mmHg), during (98.4 +/- 3.4 mmHg), or after (100.2 +/- 2.2 mmHg) intrasplenic infusion of ADM (9 ng min(-1)) (n = 11, P < 0.05). 4. ADM (9 ng min(-1)) caused an increase in intrasplenic microvascular pressure (11.3 +/- 0.3 (saline, n = 5) vs. 13.0 +/- 0.3 mmHg (9 ng min(-1) ADM, n = 6), P < 0.05). 5. ADM (1 x 10(-11) to 1 x 10(-6) M) induced greater vasorelaxation of isolated preconstricted splenic resistance arteries than veins (maximal relaxation: 60 +/- 0.9 (artery, n = 9) vs. 43 +/- 1.7 % (vein, n = 8), P < 0.05). L-NMMA (10(-4) M) partially inhibited the ADM-induced relaxation in splenic arteries (maximal relaxation: 38 +/- 3 (ADM + L-NMMA, n = 5) vs. 60 +/- 3 % (ADM + D-NMMA, n = 5), P < 0.05). 6. It is concluded that ADM increases fluid efflux from the splenic vasculature by differentially reducing pre- vs. post-capillary resistance, thus increasing intrasplenic microvascular pressure.