Joseph E. Kiss

Spermatogonial Stem Cell Transplantation into Rhesus Testes Regenerates Spermatogenesis Producing Functional Sperm

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a mans life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from four-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation.

Upper-extremity transplantation using a cell-based protocol to minimize immunosuppression.

Objective: To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure. Background: Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the risks and side effects of multidrug immunosuppression. We present our experience with upper-extremity transplantation under a novel, donor bone marrow (BM) cell-based treatment protocol (“Pittsburgh protocol”). Methods: Between March 2009 and September 2010, 5 patients received a bilateral hand (n = 2), a bilateral hand/forearm (n = 1), or a unilateral (n = 2) hand transplant. Patients were treated with alemtuzumab and methylprednisolone for induction, followed by tacrolimus monotherapy. On day 14, patients received an infusion of donor BM cells isolated from 9 vertebral bodies. Comprehensive follow-up included functional evaluation, imaging, and immunomonitoring. Results: All patients are maintained on tacrolimus monotherapy with trough levels ranging between 4 and 12 ng/mL. Skin rejections were infrequent and reversible. Patients demonstrated sustained improvements in motor function and sensory return correlating with time after transplantation and level of amputation. Side effects included transient increase in serum creatinine, hyperglycemia managed with oral hypoglycemics, minor wound infection, and hyperuricemia but no infections. Immunomonitoring revealed transient moderate levels of donor-specific antibodies, adequate immunocompetence, and no peripheral blood chimerism. Imaging demonstrated patent vessels with only mild luminal narrowing/occlusion in 1 case. Protocol skin biopsies showed absent or minimal perivascular cellular infiltrates. Conclusions: Our data suggest that this BM cell-based treatment protocol is safe, is well tolerated, and allows upper-extremity transplantation using low-dose tacrolimus monotherapy.

Intensive plasma exchange increases a disintegrin and metalloprotease with thrombospondin motifs-13 activity and reverses organ dysfunction in children with thrombocytopenia-associated multiple organ failure.

Background:Thrombocytopenia-associated multiple organ failure (TAMOF) is a poorly understood syndrome in critically ill children. A disintegrin and metalloprotease with thrombospondin motifs (ADAMTS-13), formerly known as von Willebrand factor (VWF) cleaving protease, is decreased in adults with VWF-mediated thrombotic microangiopathy, and intensive plasma exchange (PEx) both replenishes ADAMTS-13 and improves outcome in these patients. Objectives:To determine whether: 1) critically ill children with TAMOF syndrome have decreased ADAMTS-13 activity, 2) ADAMTS-13 activity correlates with platelet counts and VWF antigen, 3) the autopsies from patients who died with reduced ADAMTS-13 activity have VWF-rich microthrombi, and 4) intensive PEx will restore ADAMTS-13 activity and facilitate organ failure resolution. Design:First study: Observational. Second study:Randomized control trial. Setting:Single center university pediatric intensive care unit. Patients:First study: thirty-seven consecutive children (17 males and 20 females; ages ranging from 9 days to 23 years) identified with ≥2 organs dysfunction were enrolled. Seventy-six percent of these children had thrombocytopenia (platelet counts <100,000/mm3). Five additional critically ill children without MOF were also enrolled. In the second study, children with severe TAMOF (platelet counts <100,000/mm3 and >3 organ failure) were randomized to PEx or standard therapy. Primary physicians and parents agreed to enrollment in 10 of the 20 eligible patients with ages ranging from 1 year to 18 years. Five patients received PEx and 5 patients received standard therapy. Results:First study: children with TAMOF (n = 28) had decreased ADAMTS-13 activity, but similar plasminogen activator inhibitor-1 activity and prothrombin time compared to children with MOF without thrombocytopenia (n = 9, p < 0.05). All non-survivors (n = 7) had TAMOF, reduced ADAMTS-13 activity, and VWF-rich microvascular thromboses at autopsy. In the second study, PEx (n = 5, median 12 days, 4–28 days) restored ADAMTS-13 activity and organ function, compared to standard therapy (n = 5, p < 0.05). Conclusions:Children with TAMOF syndrome can have VWF-mediated thrombotic microangiopathy. Similar to adult experience, PEx can replenish ADAMTS-13 activity and reverse organ failure.

Effects of hostility on platelet reactivity to psychological stress in coronary heart disease patients and in healthy controls.

Previous studies have demonstrated a potential relationship between psychological stress and platelet activation, which may serve as a link between stress and myocardial infarction (MI).However, the possibility that personality traits associated with coronary heart disease may affect platelet activation has not been adequately investigated. The effect of a laboratory stressor (Type A Structured Interview (SI) and speech task) on platelet activation was assessed in 14 stable post-MI patients and 15 age-matched healthy men, using a standardized method of measuring plasma beta-thromboglobulin (BTG) levels. BTG levels were increased after the stressor (average change = 2.0 ng/ml, p =.005). Increases in BTG with stress were related to higher SI ratings of Potential for Hostility (r =.53, p =.004) and Type A behavior (r =.43, p =.02) but not to Cook-Medley-rated hostility scores. Increases in norepinephrine levels and in diastolic blood pressure were nonsignificantly related to increases in BTG levels (ps <.10), whereas increases in epinephrine levels were unrelated. Despite ceasing aspirin and other platelet inhibitors for 10 days before testing, individuals taking platelet inhibitors before the study had less change in BTG with stress (p =.05). However, after statistical adjustment for this factor, SI ratings of Potential for Hostility were still strongly related to increases in BTG with stress (adjusted r =.56, p =.002). Contrary to expectations, healthy men tended to have greater change in BTG with stress than post-MI patients (p =.06). These results indicate that acute stress increases BTG levels and that hostility is related to greater platelet reactivity, independent of any long term effects of platelet inhibition.

Hemostatic factors according to menopausal status and use of hormone replacement therapy.

The rise in cardiovascular disease (CVD) risk after menopause may be reduced by hormone replacement therapy (HRT) although the mechanism is unclear. Because little is known about the potential role of hemostatic factors, fibrinogen level and other coagulation parameters were measured in a study on the change in CVD risk factors through the climacteric (the Healthy Women Study). Of 239 subjects measured to date, 32 taking aspirin or other medications thought to alter coagulation were excluded from analyses. Results (adjusted for age and obesity) showed that women taking HRT had lower plasma concentrations of fibrinogen and higher levels of plasminogen and factor VIIc than did postmenopausal subjects not taking HRT. Pre- as compared with postmenopausal women had lower plasma levels of fibrinogen, factor VIIc, and antithrombin III. Adjusting for cigarette smoking did not change the findings. Thus, among women aged 49 to 55, selected hemostatic measures varied (within normal ranges) by menopausal status and were altered by HRT. These findings generally support a hypothesis of hemostatic change contributing to the increase of CVD after menopause. The fact that subjects taking HRT showed no increase in fibrinogen relative to premenopausal women is consistent with an observed decreased risk of CVD among women taking HRT, while the implication of an elevation in factor VIIc among these women is uncertain.

Thrombotic Thrombocytopenic Purpura: 2012 American Society for Apheresis (ASFA) Consensus Conference on Classification, Diagnosis, Management, and Future Research

The American Society for Apheresis (ASFA) conducted a 1 day consensus conference on Thrombotic Thrombocytopenic Purpura (TTP) during its annual meeting in Atlanta, GA, on April 10, 2012. The authors of this article, a subcommittee of ASFAs Clinical Applications Committee, developed several questions with regard to definitions, classification, pathophysiology, diagnosis, management, and future research in TTP. These questions were provided to the seven invited speakers who are the experts in the field of TTP. Two moderators conducted the proceedings of the conference which was attended by more than 100 participants. After each presentation, there was an open discussion that included moderator‐selected written questions submitted by the audience. A medical writer‐generated transcript of the proceedings as well as each presentation was made available to the authors. Each summary was reviewed and approved by the respective speaker before submission of this article. The subcommittee also developed seven key questions for blinded, electronic polling conducted by the moderators to generate a consensus amongst the speakers. This article includes these presentation summaries as well as results of the electronic poll. J. Clin. Apheresis 29:148–167, 2014.

Efficacy of transfusion with granulocytes from G-CSF/dexamethasone–treated donors in neutropenic patients with infection

High-dose granulocyte transfusion therapy has been available for 20 years, yet its clinical efficacy has never been conclusively demonstrated. We report here the results of RING (Resolving Infection in Neutropenia with Granulocytes), a multicenter randomized controlled trial designed to address this question. Eligible subjects were those with neutropenia (absolute neutrophil count <500/μL) and proven/probable/presumed infection. Subjects were randomized to receive either (1) standard antimicrobial therapy or (2) standard antimicrobial therapy plus daily granulocyte transfusions from donors stimulated with granulocyte colony-stimulating factor (G-CSF) and dexamethasone. The primary end point was a composite of survival plus microbial response, at 42 days after randomization. Microbial response was determined by a blinded adjudication panel. Fifty-six subjects were randomized to the granulocyte arm and 58 to the control arm. Transfused subjects received a median of 5 transfusions. Mean transfusion dose was 54.9 × 10(9) granulocytes. Overall success rates were 42% and 43% for the granulocyte and control groups, respectively (P > .99), and 49% and 41%, respectively, for subjects who received their assigned treatments (P = .64). Success rates for granulocyte and control arms did not differ within any infection type. In a post hoc analysis, subjects who received an average dose per transfusion of ≥0.6 × 10(9) granulocytes per kilogram tended to have better outcomes than those receiving a lower dose. In conclusion, there was no overall effect of granulocyte transfusion on the primary outcome, but because enrollment was half that planned, power to detect a true beneficial effect was low. RING was registered at www.clinicaltrials.gov as #NCT00627393.

Prevalence and quantitation of parvovirus B19 DNA levels in blood donors with a sensitive polymerase chain reaction screening assay

BACKGROUND: Blood donor parvovirus B19 DNA prevalence with sensitive nucleic acid test assays has recently been demonstrated to be higher than that found with assays designed to detect high viral titers in the plasma manufacturing sector.

Thrombotic thrombocytopenic purpura: recognition and management

Thrombotic thrombocytopenic purpura is a life-threatening multisystem disorder that represents both a diagnostic and a management challenge to clinicians. Early recognition of the condition coupled with rapid institution of plasma exchange has led to a dramatic improvement in prognosis. Studies performed over the past decade have elucidated the predominant pathophysiology, stemming from a deficiency of ADAMTS13, that accounts for the widespread microvascular deposition of platelet–von Willebrand factor in many sites, including the brain, kidney, and mesenteric vessels. However, in light of the mortality rate of 10–20%, much work remains to be done to translate advances in our understanding of pathophysiology into clinical practice. Improvements in medical management using immunosuppressive and other drugs are being actively explored in clinical trials. Agents that target ADAMTS13 autoantibody production by B-cells, such as anti-CD20 monoclonal antibodies, have the potential to shorten the duration of plasma exchange treatment, reduce relapses, and transform the management of this once enigmatic disorder.

The difference between fingerstick and venous hemoglobin and hematocrit varies by sex and iron stores

BACKGROUND: Fingerstick blood samples are used to estimate donor venous hemoglobin (Hb).