Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline–revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Alphaviruses are enveloped, positive sense single-stranded RNA viruses that can cause significant human diseases ranging from arthritis to encephalitis (1–3). Alphaviruses that are associated with musculoskeletal disease (arthritogenic alphaviruses) include Chikungunya virus (CHIKV), Mayaro virus (MAYV), Ross River virus (RRV), O’nyong-nyong virus (ONNV), and others; these viruses are globally distributed and transmitted by mosquitos. Symptomatic infection by arthritogenic alphaviruses is characterized by fever, rash, myalgia, as well as both acute and chronic peripheral polyarthralgia (4, 5). The arthropathy can be debilitating and persist for months to years after infection. More severe manifestations of alphavirus disease—including encephalopathy and mortality—have been reported (6, 7). These viruses cause endemic disease as well as large, sporadic global outbreaks (8–10). Currently, there are no approved vaccines or antiviral therapies for the prevention or treatment of alphavirus infection.MAYV is an arthritogenic alphavirus that was first isolated in 1954 in Trinidad, and recent outbreaks have been reported in numerous areas of Central and South America (11, 12). The primary vectors for MAYV are Haemagogus spp. mosquitoes, which transmit the virus to primates in a sylvatic cycle. However, MAYV vector competence studies have demonstrated transmission potential in multiple Aedes and Anopheles mosquitoes (13–17). The wide range and distribution of MAYV-competent vectors underscores the risk of potential urban transmission (18) and global spread (19).The alphavirus glycoprotein is composed of heterodimers of two transmembrane subunits, E2 and E1, which mediate viral attachment and membrane fusion, respectively (20–22). The prefusion E2/E1 heterodimer forms a trimeric spike that is arranged in an icosahedral lattice on the viral particle. E2 is initially expressed as a precursor polypeptide known as p62. During virus biogenesis, p62 is processed by cellular furin to generate E2 and the peripheral E3 polypeptide. E3 remains bound to the E2/E1 heterodimer during exocytic transport and prevents premature conformational changes and membrane fusion (23, 24). The release of E3 is the final step of virus maturation and primes the glycoprotein for membrane fusion.Both E2 and E1 proteins are targets of the neutralizing antibody response. Antibody-mediated protection by neutralizing monoclonal antibodies (mAbs) has been shown against several alphaviruses (25–31). We and others have reported the isolation of potent and protective neutralizing CHIKV mAbs targeting regions of E2, such as the β-connector region and the A domain (25–27). These mAbs neutralize viral particles via multiple mechanisms, including the prevention of attachment and membrane fusion. The alphavirus receptor Mxra8 binds to regions spanning the A and B domains of E2 protein (32), and neutralizing mAbs targeting these regions can effectively disrupt virus interaction with the host receptor (33).Many of the identified, neutralizing human mAbs against alphaviruses are virus-specific and do not inhibit heterologous alphaviruses. Notably, most of these mAbs target CHIKV, and there are few examples of MAYV-reactive human mAbs. Recent work has demonstrated the cross-reactivity and cross-neutralization of human polyclonal sera to heterologous alphaviruses (34–36), suggesting that broadly reactive and/or broadly neutralizing monoclonal antibodies (bNAbs) may be elicited by alphavirus infection in humans. While a number of murine bNAbs have been characterized (30, 37, 38), few human bNAbs that engage multiple alphaviruses have been described (33). For example, the murine mAb CHK-265 can protect against CHIKV, MAYV, and RRV challenge in mice (38). More recently, a human mAb, RRV-12, was shown to protect mice against RRV and MAYV infection (33). Both CHK-265 and RRV-12 broadly neutralize infection by engaging the B domain of E2, but whether such protective alphavirus bNAbs are elicited commonly during the course of human CHIKV infection is unknown.Here, we describe the isolation and characterization of cross-reactive alphavirus mAbs from a CHIKV-convalescent donor. We employed a single B cell sorting strategy using a heterologous MAYV antigen to isolate 33 cross-reactive mAbs and found that they target multiple epitopes on the E1 and E2 proteins. We identified five human bNAbs that neutralize CHIKV, MAYV, and other alphaviruses with differing potencies. Epitope binning and viral escape studies suggest that human bNAbs target related but distinct regions of the B domain of E2. Remarkably, the sequence analysis of human bNAbs showed few somatic mutations, and inferred germline variants largely retained neutralizing function. Two bNAbs demonstrated protection against both CHIKV- and MAYV-induced musculoskeletal disease in mice. Together, these studies further define heterologous humoral immunity among related alphaviruses in humans as well as the determinants of antibody-mediated cross-protection.     

Comparative efficacy of desferrioxamine, deferiprone and in combination on iron chelation in thalassemic children

OBJECTIVE: Ascertainment of an appropriate strategy of iron chelation for multi-transfused thalassemic children in developing countries. DESIGN: Prospective study from May 2000 to April 2001. SETTING: Urban tertiary care center. METHODS: Thirty thalassemic children having received more than 20 blood transfusions and a serum ferritin greater than 1500 ng/ml were enrolled and randomized into three groups. Group I received desferrioxamine (DFX) at a dose of 40 mg/kg subcutaneously, 5 days/week. Children in group II received oral deferiprone (L1) at a dose of 75 mg/kg/day daily and group III received a combination of daily L1 at a dose of 75 mg/kg/day and DFX at a dose of 40 mg/kg/day two times per week. The assessment of chelation was done by 24-hr urinary iron excretion (UIE) and measurement of serum ferritin levels at start and after 6 months of follow up. Statistical difference of serum ferritin levels between the three groups was assessed by applying analysis of variance. Analysis of covariance was applied to find out the urinary iron excretion keeping serum ferritin values same in each groups. RESULTS: Ferritin levels after 6 months of intervention were maximally decreased in group I. There was a significant difference between groups I and II however, no difference was noted between groups I and group III. There was no statistically significant difference in mean urinary iron excretion by keeping the initial serum ferritin levels equal though it was found to be more in group III as compared to other groups. CONCLUSIONS: DFX is the most effective chelating drug in iron overloaded multi-transfused thalassemic patients. In view of cost and unacceptability of daily DFX injections, combination therapy is an effective method of chelation thus increasing the compliance and cost effectiveness. Deferiprone (L1) alone is not an effective mode of chelation when used for a short period.     

End stage renal disease due to bilateral renal malakoplakia.

Malakoplakia typically affects the bladders of immunocompromised adults who have defective intracellular killing of Escherichia coli. Renal malakoplakia is rare in children and generally has a good outcome. In the case presented, however, it caused end stage renal failure in a 5 year old girl. The management dilemmas surrounding renal transplantation are highlighted.     

Parotid area sign: a clinical test for the diagnosis of fluid overload in hysteroscopic surgery

STUDY OBJECTIVE: To describe the clinical test parotid area sign, which is used to assess fluid absorption during resectoscopic surgery and to compare the test with volumetric fluid balance method with respect to its ability to detect fluid overload. DESIGN: Historical cohort study (Canadian Task Force classification II-1). SETTING: Tertiary endoscopy center. PATIENTS: Eighty-six women who underwent resectoscopic surgery between 1999 and 2004 at our center. INTERVENTION: The volumetric fluid balance method was used to evaluate glycine absorption (glycine deficit) during the surgery. A flexometallic ruler was placed on the left cheek of the patient between 2 fixed points: the midpoint of the philtrum and a point on the mastoid prominence, and this distance (philtrum-mastoid prominence distance) was measured at the beginning of every 3 minutes during, and at the end of the procedure. MEASUREMENTS AND MAIN RESULTS: Eighty-six patients were divided into 2 groups: Group A, which included patients with absorption less than 1000 mL as measured by the volumetric method; and Group B, which included patients with absorption of 1000 mL or more. The results of the parotid area sign test in the 2 groups were compared. The 2 groups were comparable with respect to the age, weight, preoperative measured philtrum-mastoid prominence distance, and hospital stay. The median (and average absolute deviation) operating time in group A (15 minutes [and 6.79]; range 8-60 minutes; 95% CI of the median, 15-20 minutes) was significantly lower than the median (and average absolute deviation) operating time in group B (25 minutes [and 8.96]; range 9-60 minutes; 95% CI of the median, 20-25 minutes; p <.001). The mean postoperative philtrum-mastoid prominence distance measured in patients of group A (14.23 +/- 0.396 cm [range 14-16 cm, 95% CI 14.10-14.36 cm]) was significantly lower than that in group B [14.76 +/- 0.622 cm (range 14-17 cm, 95% CI 14.58-15.12 cm]; p <.001). By paired t test, the change in the philtrum-mastoid prominence distance after surgery as compared with the value before surgery in each patient was found to be insignificant in group A (p =.86). However, it was found to be significant in group B (p <.001). The increase in the measured philtrum-mastoid prominence distance (i.e., postoperative measurement minus the preoperative measurement) in each patient after surgery was significantly more in group B (mean +/- SD, 0.54 +/- 0.362 cm [range 0-2 cm, 95% CI 0.43-0.65 cm]) than that in group A (mean +/- SD, 0.03 +/- 0.091 cm [range 0-0.4 cm, 95% CI 0.008-0.06 cm]; p <.001). The correlation coefficient for the increase in the philtrum-mastoid prominence distance as the glycine deficit increased in the 2 groups considered together was significant (r = 0.937, p <.01). The partial regression coefficient b value for the effect of duration of surgery while controlling for the effect of fluid deficit was 0.008 (p <.001), and the b value for the effect of fluid deficit while controlling for the effect of duration of surgery was 0.437 (p <.001). The regression coefficient r value (0.727) for the goodness of the fit of the regression line to the data sets was also significant (p <.001). The sensitivity of the test with respect to the volumetric fluid balance is 97.8% (95% CI, 87.28%-99.88%) and specificity is 92.3% (95% CI, 78.03%-97.99%). The negative predictive value is 97.30% (95% CI, 84.19%-99.85%) and positive predictive value is 93.87 (95% CI, 82.13%-98.40%). The conventional positive likelihood ratio for the test is 12.72 (95% CI 4.28-37.77). The conventional negative likelihood ratio is 0.023 (95% CI 0.003-0.16). CONCLUSION: The parotid area sign is a simple, effective, and easy-to-perform test (in real time continuously) that requires minimal equipment or training. It supplements the volumetric fluid balance method in the detection of fluid overload (1.5% glycine) during resectoscopic surgery. It may also enable us to detect fluid overload when volumetric fluid balance method fails to detect extraneous losses caused by spillage.     

Amyloid β and neuromelanin—Toxic or protective molecules?: The cellular context makes the difference

Alzheimer's disease (AD) and Parkinson's disease (PD) share several pathological mechanisms. The parallels between amyloid beta (Aβ) in AD and -synuclein in PD have been discussed in several reports. However, studies of the last few years show that Aβ also shares several important characteristics with neuromelanin (NM), whose role in PD is emerging. First, both molecules accumulate with aging, the greatest risk factor for AD and PD. Second, in spite of their different structures, Aβ and NM have similar characteristics that could also lead to neuroprotection. Metals are required to catalyze their formation and they can bind large amounts of these metals, generating stable complexes and thus playing a protective role against metal toxicity. Moreover, they may be able to remove toxic species such as oligopeptides and excess cytosolic dopamine. Third, both Aβ and NM have been implicated in parallel aspects of the neuronal death that underlies AD and PD, respectively. For example, both molecules can activate microglia, inducing release of toxic factors such as tumor necrosis factor- (TNF-), interleukin-6 (IL-6), and nitric oxide (NO). A careful analysis of these parallel effects of Aβ and NM, including their seemingly paradoxical ability to participate in both cell death and protection, may lead to an improved understanding of the roles of these molecules in neurodegeneration and also provide insights into possible parallels in the pathological mechanisms underlying AD and PD.     

Propionibacterium skull osteomyelitis treated with daptomycin

Propionibacterium acnes (P. acnes) is emerging as a pathogen in postneurosurgical infections. A case of cranial bone flap infection due to P. acnes successfully treated with daptomycin is presented. This case demonstrates the potential of daptomycin as an option in the treatment of P. acnes.     

Enhanced neuronal loss under perinatal hypothyroidism involves impaired neurotrophic signaling and increased proteolysis of p75NTR

Recognition of the molecular events that lead to enhanced cell death is vital to understand the developmental cerebellar defects under hypothyroidism. Though neurotrophins promote the survival and development of neurons in the cerebellum, but the mechanism of their insufficiency mediated cell loss under hypothyroidism is unknown. Here in developmental hypothyroid rat model we report that hypothyroidism induced neuronal loss involve down regulation of neurotrophic survival signaling and increased truncation of the receptor p75^NTR. Results showed that perinatal hypothyroidism besides repressing the expression of BDNF also impairs the maturation of NGF which results in decreased activation of ERK, CREB, NF-kappaB and AKT. Furthermore hypothyroidism caused an enhanced expression and proteolysis of p75^NTR. The increased proteolysis of p75^NTR in vivo and its association with death of granule neurons brings forward hitherto a p75^NTR dependence signaling which along with compromised survival signaling could provide a neurotrophic basis of understanding the cause of enhanced cell death in developing cerebellum under hypothyroidism.     

Bilateral Hahn–Steinthal fracture: a case report and review of literature

Capitellar fractures are rare. If anatomy is not reconstructed accurately, elbow function is sub-optimal. Various studies have shown good outcome in Type I Hahn–Steinthal fractures. There is only one report of a bilateral capitellar fracture in the English literature so far. We report another case of the same. A 26-year-old mother slipped and fell on level ground. She sustained closed injury to both her elbows. Radiographs revealed bilateral, displaced Hahn–Steinthal fracture of the capitellum. She underwent open anatomic reduction and stabilisation with two partially threaded 4 mm cancellous screws, inserted from posterior to anterior. At 2 months, both fractures united. At final follow-up, 19 months post-operatively, she had full range of movement of both elbows. Anatomic reduction, stable internal fixation and early mobilisation of Type I Hahn–Steinthal capitellar fracture achieves excellent results.