Table_1_Nanobodies: a promising approach to treatment of viral diseases.docx

Since their discovery in the 1990s, heavy chain antibodies have garnered significant interest in the scientific community. These antibodies, found in camelids such as llamas and alpacas, exhibit distinct characteristics from conventional antibodies due to the absence of a light chain in their structure. Furthermore, they possess a single antigen-binding domain known as VHH or Nanobody (Nb). With a small size of approximately 15 kDa, these Nbs demonstrate improved characteristics compared to conventional antibodies, including greater physicochemical stability and enhanced biodistribution, enabling them to bind inaccessible epitopes more effectively. As a result, Nbs have found numerous applications in various medical and veterinary fields, particularly in diagnostics and therapeutics. Advances in biotechnology have made the production of recombinant antibodies feasible and compatible with large-scale manufacturing. Through the construction of immune phage libraries that display VHHs and subsequent selection through biopanning, it has become possible to isolate specific Nbs targeting pharmaceutical targets of interest, such as viruses. This review describes the processes involved in nanobody production, from hyperimmunization to purification, with the aim of their application in the pharmaceutical industry.</p

Neurotrophic factor expression (BDNF and GDNF) by RT-qPCR in the ventral horn spinal cord 1 and 4 weeks after avulsion.

<p>Expression of mean ratio of the ipsi-/contralateral mRNA for BDNF obtained by RT-qPCR in the lumbar spinal cord one (A) and four (B) weeks after avulsion. Note that one week after avulsion AV+S+HC enhanced the BDNF mRNA production compared to others groups (*p<0.05, n = 5). Expression of mean ratio of the ipsi-/contralateral mRNA for GDNF obtained by RT-qPCR in the lumbar spinal cord one (C) and four (D) weeks after avulsion. Note that, one week after avulsion in the AV+S+HC group, there is enhanced GDNF mRNA production as compared to others groups (*p<0.05, n = 5).</p

Immunohistochemical analysis of the spinal cord ventral horn stained with anti-synaptophysin 4 and 8 weeks after VRA.

<p>Observe the preservation of synaptophysin labeling, especially at the surface of the lesioned motoneurons in implanted group and both implanted with mononuclear cells treatment. After 4 weeks post lesion (A, C, E and G) and after 8 weeks (B, D, F and H). (A and B) AV, (C and D) AV+S, (E and F) AV+S+HC and (G and H) AV+S+IC. Scale bar  = 50 µm. (I) Quantification of synaptic covering obtained by the ratio ipsi/contralateral sides of the integrated density of pixels at lamina IX four and eight weeks after injury. (†† p<0.01 comparing all groups 4 weeks after avulsion, § p<0.05 comparing all groups 8 weeks after AV and * p<0.05 comparing AV+S+IC at two different times after avulsion, n = 5).</p

Microglial analysis of the spinal cord ventral horn 4 and 8 weeks after VRA.

<p>The quantification analysis on motoneuron cell bodies stained with anti- Iba1 of the ipsilateral side of VRA after 4 weeks (A, C, E and G) and after 8 weeks (B, D, F and H). (A and B) AV, (C and D) AV+S, (E and F) AV+S+HC and (G and H) AV+S+IC. Scale bar  = 50 µm. (I) The mean ratio of the ipsil-/contralateral integrated intensity of pixels of the ipsilateral and contralateral sides in both groups. Note the decrease in microglial reactivity in AV+S and AV+S+IC as compared 4 and 8 weeks after injury. (J) There were significant increase in the synthesis of Iba-1 mRNA in the lumbar spinal cord one week after avulsion (*p<0.05 and **p<0.01, n = 5). (K) The RT-qPCR, performed 4 weeks after avulsion, did not demonstrate significant differences between groups in Iba-1 mRNA.</p

Nissl-stained spinal cord transverse sections at lamina IX illustrating the neuroprotective effects of root reimplantation and MC treatment on motoneurons 4 and 8 weeks after VRA.

<p>Motoneuron cell bodies of the ipsilateral side of VRA after 4 weeks (A, C, E and G) and after 8 weeks (B, D, F and H). (A and B) AV, (C and D) AV+S, (E and F) AV+S+HC and (G and H) AV+S+IC. Scale bar  = 50 µm. (I) Percentage of neuronal survival after ventral root avulsion, reimplantation and reimplantantion with MC. Note a significant rescue of lesioned neurons in the implanted groups with and without cells in two different survival times (4 and 8 weeks). This neuroprotection was even more intense in AV+S+HC 4 weeks after avulsion. (†† p<0.01 and ††† p<0.001 comparing all groups 4 weeks after avulsion, § p<0.05, §§ p<0.01 and §§§ p<0.001 comparing all groups 8 weeks after AV and *p<0.05 comparing AV+S+HC at two different times after avulsion, n = 5).</p

Additional file 1: of Heterologous fibrin sealant derived from snake venom: from bench to bedside – an overview

The video shows a six-min overview of the production and application of the fibrin sealant derived from snake venom and buffalo blood (available at https://youtu.be/y6ho6M0amA8 ). (DOCX 11 kb

Glial fibrillary acidic protein (GFAP) in the spinal cord ventral horn.

<p>Immunohistochemical analysis of the anterior horn of the spinal cord was labeled with anti-GFAP, 4 and 8 weeks after injury to assess the degree of astroglial reactivity after root avulsion (A–H). Representative images of AV, AV+S, AV+S+HC and AV+S+IC. Scale bar  = 50 µm. Observe that ventral root implantation and cell treatment did not increase astroglial reaction. (I) The mean ratio of the ipsi-/contralateral integrated intensity of pixels of the ipsilateral and contralateral sides in all groups. (* p<0.05, n = 5). (J) One week after avulsion, there were no differences between groups by GFAPmRNA analysis. (K) The RT-qPCR performed 4 weeks after avulsion demonstrated significant decrease in the synthesis of GFAP mRNA in AV+S group compared with AV (*p<0.05, n = 5).</p

Motor function recovery after ventral root treatment.

<p>(A) Graph of the peroneal nerve functional index up to 8 weeks after avulsion. There is a significantly better performance of the three implanted groups compared to AV from the first week post lesion until the eighth week (***p<0.001, **p<0.01 and *p<0.05 n = 10). (B) Restoration of weight-bearing capacity following avulsion. There is also a restoration of weight-bearing capacity following avulsion in implanted groups with or without cells from the first up to the eight week after injury. Values are expressed as the ratio of ipsi-/contralateral pressure exerted by the paw on the catwalk platform (***p<0.001 and *p<0.05, n = 10).</p

Iba1 immunolabeling in the spinal cord ventral horn.

<p>Immunohistochemical analysis of the anterior horn of the spinal cord was labeled with anti-Iba1 12 weeks after injury to assess the degree of microglial reactivity after root avulsion. (A and B) Normal immunolabeling of Iba1 on the contralateral side. (C) Ipsilateral side of the lesion in avulsion and (D) reimplantation after avulsion. Scale bar = 50 µm. (E) The mean ratio of the ipsil-/contralateral integrated intensity of pixels of the ipsilateral and contralateral sides in both groups. No significant differences between groups were observed (n = 5).</p

Paw pressure evaluation with the Catwalk system.

<p>The walking track test apparatus: (A) CatWalk machine and (B) an example of a rat at the walkway and the green plantar impression (arrow). In (C), observe a rat from the implanted group using the right paw 12 weeks after injury, whereas the avulsed rat without root repair cannot control the lesioned limb (D), dotted circle indicates the place where the paralyzed paw was supposed to be seen.</p